Methods of treatment using cysteine variants of interleukin-11

ABSTRACT

Disclosed are cysteine variants of interleukin-11 (IL-11) and methods of making and using such proteins in therapeutic applications.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of U.S. application Ser. No.12/391,896, filed Feb. 24, 2009, now U.S. Pat. No. 8,133,480, which is aContinuation of U.S. application Ser. No. 11/544,473, filed Oct. 5,2006, now U.S. Pat. No. 7,495,087, which claims the benefit of priorityunder 35 U.S.C. §119(e) from U.S. Provisional Application No.60/724,204, filed Oct. 5, 2005. U.S. application Ser. No. 11/544,473 isalso a continuation-in-part of U.S. application Ser. No. 10/298,148,filed Nov. 15, 2002, now U.S. Pat. No. 7,153,943, which claims thebenefit of priority under 35 U.S.C. §119(e) from U.S. ProvisionalApplication Ser. No. 60/332,285, filed Nov. 15, 2001, and from U.S.Provisional Application Ser. No. 60/418,040, filed Oct. 11, 2002. U.S.application Ser. No. 10/298,148 is a continuation-in-part of U.S.application Ser. No. 09/462,941, filed Jan. 14, 2000, now U.S. Pat. No.6,608,183, which is a national stage filing under 35 U.S.C. 371 of PCTApplication Serial No. PCT/US98/14497, filed Jul. 13, 1998, and whichclaims the benefit of priority from U.S. Provisional Application Ser.No. 60/052,516, filed Jul. 14, 1997. The entire disclosure of each ofthe above-identified applications is incorporated herein by reference.

GOVERNMENT SUPPORT

This invention was made in part with government support under NIH GrantNo. 1R43 CA084851 and NIH Grant No. 2R44CA084851, each awarded by theNational Institutes of Health. The government has certain rights to thisinvention.

REFERENCE TO SEQUENCE LISTING

This application contains a Sequence Listing submitted on a compactdisc, in duplicate. Each of the two compact discs, which are identicalto each other pursuant to 37 CFR §1.52(e)(4), contains the followingfile: “4152-1-PUS-14.ST25.txt”, having a size in bytes of 45 KB,recorded on 5 Oct. 2006. The information contained on the compact discis hereby incorporated by reference in its entirety pursuant to 37 CFR§1.77(b)(4).

FIELD OF THE INVENTION

The present invention relates to genetically engineered therapeuticproteins and methods of use thereof. More specifically, the engineeredproteins include cysteine variants of the growth hormone supergenefamily, such as interleukin-11 (IL-11) and related proteins, and methodsof making and using such proteins.

BACKGROUND OF THE INVENTION

The following proteins are encoded by genes of the growth hormone (GH)supergene family (Bazan (1990); Mott and Campbell (1995); Silvennoinenand Ihle (1996)): growth hormone, prolactin, placental lactogen,erythropoietin (EPO), thrombopoietin (TPO), interleukin-2 (IL-2), IL-3,IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-11, IL-12 (p35 subunit), IL-13,IL-15, oncostatin M, ciliary neurotrophic factor, leukemia inhibitoryfactor, alpha interferon, beta interferon, gamma interferon, omegainterferon, tau interferon, granulocyte-colony stimulating factor(G-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF),macrophage colony stimulating factor (M-CSF) and cardiotrophin-1 (CT-1)(“the GH supergene family”). It is anticipated that additional membersof this gene family will be identified in the future through genecloning and sequencing. Members of the GH supergene family have similarsecondary and tertiary structures, despite the fact that they generallyhave limited amino acid or DNA sequence identity. The shared structuralfeatures allow new members of the gene family to be readily identified.

There is considerable interest on the part of patients and healthcareproviders in the development of long acting, “user-friendly” proteintherapeutics. Proteins are expensive to manufacture and, unlikeconventional small molecule drugs, are not readily absorbed by the body.Moreover, they are digested if taken orally. Therefore, natural proteinsmust be administered by injection. After injection, most proteins arecleared rapidly from the body, necessitating frequent, often daily,injections. Patients dislike injections, which leads to reducedcompliance and reduced drug efficacy. Some proteins, such aserythropoietin (EPO), are effective when administered less often (threetimes per week for EPO) because they are glycosylated. However,glycosylated proteins are produced using expensive mammalian cellexpression systems.

The length of time an injected protein remains in the body is finite andis determined by, e.g., the protein's size and whether or not theprotein contains covalent modifications such as glycosylation.Circulating concentrations of injected proteins change constantly, oftenby several orders of magnitude, over a 24-hour period. Rapidly changingconcentrations of protein agonists can have dramatic downstreamconsequences, at times under-stimulating and at other timesover-stimulating target cells. Similar problems plague proteinantagonists. These fluctuations can lead to decreased efficacy andincreased frequency of adverse side effects for protein therapeutics.The rapid clearance of recombinant proteins from the body significantlyincreases the amount of protein required per patient and dramaticallyincreases the cost of treatment. The cost of human proteinpharmaceuticals is expected to increase dramatically in the years aheadas new and existing drugs are approved for more disease indications.Thus, there is a need to develop protein technologies that improve theefficacy of protein therapeutics, lessen the need for frequent delivery,and lower the costs of protein therapeutics to patients and healthcareproviders.

SUMMARY OF THE INVENTION

One embodiment of the present invention relates to a method to treat ananimal with a disease or condition that can be treated by wild-typeinterleukin-11 (IL-11), to stimulate platelet production in an animal,or to accelerate an animal's recovery from thrombocytopenia. The methodincludes administering to the animal an interleukin-11 (IL-11) cysteinemutein, wherein the mutein has biological activity in vitro as measuredby proliferation of a cell line that proliferates in response to IL-11.The thrombocytopenia can include, but is not limited to: (a)thrombocytopenia resulting from myelosuppressive chemotherapy; (b)thrombocytopenia resulting from other chemical treatments; (c)thrombocytopenia resulting from radiological treatments; (d)thrombocytopenia resulting from disease; (e) thrombocytopenia resultingfrom idiopathic causes; (f) thrombocytopenia resulting from drugtreatments, including interferons and ribavarin; (g) thrombocytopenia inneonates; (h) thrombocytopenia resulting from myelodysplastic syndromes;(i) thrombocytopenia resulting from aplastic anemia; and (j)thrombocytopenia resulting from cirrhosis. In one aspect, thethrombocytopenia is thrombocytopenia resulting from myelosuppressivechemotherapy.

In one aspect of this embodiment, the IL-11 cysteine mutein comprises atleast one non-native cysteine residue which has been added, either bysubstitution for an amino acid in the natural protein sequence or byinsertion between two adjacent amino acids in the natural proteinsequence, in a region of the protein selected from: the A-B loop, theB-C loop, the C-D loop, the first three or last three amino acids inhelix A, the first three or last three amino acids in helix B, the firstthree or last three amino acids in helix C, the first three or lastthree amino acids in helix D, the amino acids preceding helix A, and theamino acids following helix D, wherein the mutein has biologicalactivity in vitro as measured by proliferation of a cell line thatproliferates in response to IL-11. In one aspect of this embodiment, theIL-11 cysteine mutein comprises at least one non-native cysteine residuewhich has been added preceding the N-terminal amino acid of the matureprotein or following the C-terminal amino acid of the protein, whereinthe mutein has biological activity in vitro as measured by proliferationof a cell line that proliferates in response to IL-11. In one aspect,the cysteine mutein comprises at least one cysteine residue substitutedfor an amino acid in wild-type IL-11 (SEQ ID NO:17) at a positionselected from: any of positions 22-36, any of positions 37-39, any ofpositions 54-56, any of positions 57-91, any of positions 92-94, any ofpositions 110-112, any of positions 113-124, any of positions 125-127,any of positions 145-147, any of positions 148-172, any of positions173-175, any of positions 194-196, and any of positions 197-199, whereinthe mutein has biological activity in vitro as measured by proliferationof a cell line that proliferates in response to IL-11. In anotheraspect, the cysteine mutein comprises at least one cysteine residuesubstituted for an amino acid selected in SEQ ID NO:17 from: P22, G23,P24, P25, P26, G27, E38, L39, D69, L72, S74, T77, A114, S117, E123,A148, Q151, A158, A162, and S165, wherein the mutein has biologicalactivity in vitro as measured by proliferation of a cell line thatproliferates in response to IL-11. In another aspect, the cysteinemutein comprises at least two cysteine substitutions, wherein a cysteineresidue is substituted for an amino acid in SEQ ID NO:17 selected from:P25 and T77, P25 and S117, P25 and S165, P24 and P25, D69 and T77, andA162 and S165, wherein the mutein has biological activity in vitro asmeasured by proliferation of a cell line that proliferates in responseto IL-11.

In one aspect of this embodiment, the cysteine mutein is modified withat least one polyethylene glycol. In another aspect, the IL-11 cysteinemutein is modified with a cysteine-reactive moiety, including, but notlimited to, polyethylene glycol.

The cysteine mutein can be administered by a route including, but notlimited to, intravenous administration, intraperitoneal administration,intramuscular administration, intranodal administration, intracoronaryadministration, intraarterial administration, subcutaneousadministration, transdermal delivery, intratracheal administration,intraarticular administration, intraventricular administration,inhalation, intranasal, intracranial, intraspinal, intraocular, aural,oral, pulmonary administration, impregnation of a catheter, and directinjection into a tissue.

Another embodiment of the invention relates to a cysteine mutein ofinterleukin-11 (IL-11) of SEQ ID NO:17, wherein a cysteine residue issubstituted for at least one amino acid selected from: P22, G23, P24,P25, P26, G27, E38, L39, D69, L72, S74, T77, A114, S117, E123, A148,Q151, A158, A162, and S165, wherein the mutein has biological activityin vitro as measured by proliferation of a cell line that proliferatesin response to IL-11. In one aspect of this embodiment, the cysteinemutein comprises at least two cysteine substitutions, wherein a cysteineresidue is substituted for an amino acid in SEQ ID NO:17 selected from:P25 and T77, P25 and S117, P25 and S165, P24 and P25, D69 and T77, andA162 and S165, and wherein the mutein has biological activity in vitroas measured by proliferation of a cell line that proliferates inresponse to IL-11. In another aspect, the substituted cysteine residueis modified with at least one polyethylene glycol. In one aspect, thecysteine mutein is modified with a cysteine-reactive moiety, including,but not limited to, polyethylene glycol.

Another embodiment of the present invention relates to a compositionincluding any of the IL-11 cysteine variants described herein, and apharmaceutically acceptable carrier.

Another embodiment of the present invention relates to a method tostimulate platelet production in an animal, or to accelerate an animal'srecovery from thrombocytopenia, which includes administering to theanimal any of the IL-11 cysteine muteins described above.

Yet another embodiment of the present invention relates to a method toproduce a cysteine mutein of IL-11. Such a method includes the steps ofproduction and purification of the IL-11 mutein using an insect systemas described in any of Examples 2-4, using an E. coli system asdescribed in Examples 7-8, or using an E. coli intein system asdescribed in Example 9.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a solution to problems associated withprotein therapeutics by providing methods to prolong the circulatinghalf-lives of protein therapeutics in the body so that the proteins donot have to be injected frequently. This solution also satisfies theneeds and desires of patients for protein therapeutics that are“user-friendly”, i.e., protein therapeutics that do not require frequentinjections. The present invention solves these and other problems byproviding biologically active, cysteine-added variants of members of thegrowth hormone supergene family, and in particular, interleukin-11(IL-11). The invention also provides for the chemical modification ofthese variants with cysteine-reactive polymers or other types ofcysteine-reactive moieties to produce derivatives thereof and themolecules so produced. The invention also provides for therapeuticmethods using the protein variants described herein.

Accordingly, the present invention relates to cysteine variants andparticularly, cysteine variants of IL-11, and, among other things, thesite-specific conjugation of such proteins with polyethylene glycol(PEG) or other such moieties. PEG is a non-antigenic, inert polymer thatsignificantly prolongs the length of time a protein circulates in thebody. This allows the protein to be effective for a longer period oftime. Covalent modification of proteins with PEG has proven to be auseful method to extend the circulating half-lives of proteins in thebody (Abuchowski et al., 1984; Hershfield, 1987; Meyers et al., 1991).Covalent attachment of PEG to a protein increases the protein'seffective size and reduces its rate of clearance rate from the body.PEGs are commercially available in several sizes, allowing thecirculating half-lives of PEG-modified proteins to be tailored forindividual indications through use of different size PEGs. Otherbenefits of PEG modification include an increase in protein solubility,an increase in in vivo protein stability and a decrease in proteinimmunogenicity (Katre et al., 1987; Katre, 1990).

A preferred method for PEGylating proteins is to covalently attach PEGto cysteine residues using cysteine-reactive PEGs. A number of highlyspecific, cysteine-reactive PEGs with different reactive groups (e.g.,maleimide, vinylsulfone) and different size PEGs (2-20 kDa) arecommercially available (e.g., from Shearwater, Polymers, Inc.,Huntsville, Ala.). At neutral pH, these PEG reagents selectively attachto “free” cysteine residues, i.e., cysteine residues not involved indisulfide bonds. The conjugates are hydrolytically stable. Use ofcysteine-reactive PEGs allows the development of homogeneous PEG-proteinconjugates of defined structure.

Considerable progress has been made in recent years in determining thestructures of commercially important protein therapeutics andunderstanding how they interact with their protein targets, e.g.,cell-surface receptors, proteases, etc. This structural information canbe used to design PEG-protein conjugates using cysteine-reactive PEGs.Cysteine residues in most proteins participate in disulfide bonds andare not available for PEGylation using cysteine-reactive PEGs. Throughin vitro mutagenesis using recombinant DNA techniques, additionalcysteine residues can be introduced anywhere into the protein. The addedcysteines can be introduced at the beginning of the protein, at the endof the protein, between two amino acids in the protein sequence or,preferably, substituted for an existing amino acid in the proteinsequence. The newly added “free” cysteines can serve as sites for thespecific attachment of a PEG molecule using cysteine-reactive PEGs. Theadded cysteine must be exposed on the protein's surface and accessiblefor PEGylation for this method to be successful. If the site used tointroduce an added cysteine site is non-essential for biologicalactivity, then the PEGylated protein will display essentially wild type(normal) in vitro bioactivity. The major technical challenge inPEGylating proteins with cysteine-reactive PEGs is the identification ofsurface exposed, non-essential regions in the target protein wherecysteine residues can be added or substituted for existing amino acidswithout loss of bioactivity.

Cysteine-added variants of a few human proteins and PEG-polymerconjugates of these proteins have been described. U.S. Pat. No.5,206,344 describes cysteine-added variants of IL-2. Thesecysteine-added variants are located within the first 20 amino acids fromthe amino terminus of the mature IL-2 polypeptide chain. The preferredcysteine variant is at position 3 of the mature polypeptide chain, whichcorresponds to a threonine residue that is O-glycosylated in thenaturally occurring protein. Substitution of cysteine for threonine atposition 3 yields an IL-2 variant that can be PEGylated with acysteine-reactive PEG and retain full in vitro bioactivity (Goodson andKatre, 1990). In contrast, natural IL-2 PEGylated with lysine-reactivePEGs displays reduced in vitro bioactivity (Goodson and Katre, 1990).The effects of cysteine substitutions at other positions in IL-2 werenot reported.

U.S. Pat. No. 5,166,322 teaches cysteine-added variants of IL-3. Thesevariants are located within the first 14 amino acids from the N-terminusof the mature protein sequence. The patent teaches expression of theproteins in bacteria and covalent modification of the proteins withcysteine-reactive PEGs. No information is provided as to whether thecysteine-added variants and PEG-conjugates of IL-3 are biologicallyactive. Cysteine-added variants at other positions in the polypeptidechain were not reported.

PCT Patent Publication No. WO 9412219 and PCT Patent Application No.PCT/US95/06540 teach cysteine-added variants of insulin-like growthfactor-I (IGF-I). IGF-I has a very different structure from GH and isnot a member of the GH supergene family (Mott and Campbell, 1995).Cysteine substitutions at many positions in the IGF-I protein aredescribed. Only certain of the cysteine-added variants are biologicallyactive. The preferred site for the cysteine added variant is at aminoacid position 69 in the mature protein chain. Cysteine substitutions atpositions near the N-terminus of the protein (residues 1-3) yieldedIGF-I variants with reduced biological activities and improper disulfidebonds.

PCT Patent Publication No. WO 94/22466 teaches two cysteine-addedvariants of insulin-like growth factor (IGF) binding protein-1, whichhas a very different structure than GH and is not a member of the GHsupergene family. The two cysteine-added IGF binding protein-1 variantsdisclosed are located at positions 98 and 101 in the mature proteinchain and correspond to serine residues that are phosphorylated in thenaturally-occurring protein.

U.S. patent application Ser. No. 07/822,296 teaches cysteine addedvariants of tumor necrosis factor binding protein, which is a soluble,truncated form of the tumor necrosis factor cellular receptor. Tumornecrosis factor binding protein has a very different structure than GHand is not a member of the GH supergene family.

IGF-I, IGF binding protein-1 and tumor necrosis factor binding proteinhave secondary and tertiary structures that are very different from GHand the proteins are not members of the GH supergene family. Because ofthis, it is difficult to use the information gained from studies ofIGF-I, IGF binding protein-1 and tumor necrosis factor binding proteinto create cysteine-added variants of members of the GH supergene family.The studies with IL-2 and IL-3 were carried out before the structures ofIL-2 and IL-3 were known (McKay 1992; Bazan, 1992) and before it wasknown that these proteins are members of the GH supergene family.Previous experiments aimed at identifying preferred sites for addingcysteine residues to IL-2 and IL-3 were largely empirical and wereperformed prior to experiments indicating that members of the GHsupergene family possessed similar secondary and tertiary structures.

Based on the structural information now available for members of the GHsupergene family, the present invention provides “rules” for determininga priori which regions and amino acid residues in members of the GHsupergene family can be used to introduce or substitute cysteineresidues without significant loss of biological activity. In contrast tothe naturally occurring proteins, these cysteine-added variants ofmembers of the GH supergene family will possess novel properties such asthe ability to be covalently modified at defined sites within thepolypeptide chain with cysteine-reactive polymers or other types ofcysteine-reactive moieties. The covalently modified proteins will bebiologically active.

GH is the best-studied member of the GH supergene family. GH is a 22 kDaprotein secreted by the pituitary gland. GH stimulates metabolism ofbone, cartilage and muscle and is the body's primary hormone forstimulating somatic growth during childhood. Recombinant human GH (rhGH)is used to treat short stature resulting from GH inadequacy and renalfailure in children. GH is not glycosylated and can be produced in afully active form in bacteria. The protein has a short in vivo half-lifeand must be administered by daily subcutaneous injection for maximumeffectiveness (MacGillivray et al., 1996). Recombinant human GH (rhGH)was approved recently for treating cachexia in AIDS patients and isunder study for treating cachexia associated with other diseases.

The sequence of human GH is well known (see, e.g., Martial et al. 1979;Goeddel et al. 1979 which are incorporated herein by reference; SEQ IDNO:1). GH is closely related in sequence to prolactin and placentallactogen and these three proteins were considered originally to comprisea small gene family. The primary sequence of GH is highly conservedamong animal species (Abdel-Meguid et al., 1987), consistent with theprotein's broad species cross-reactivity. The three dimensional foldingpattern of porcine GH has been solved by X-ray crystallography(Abdel-Meguid et al., 1987). The protein has a compact globularstructure, comprising four amphipathic alpha helical bundles joined byloops. Human GH has a similar structure (de Vos et al., 1992). The fouralpha helical regions are termed A-D beginning from the N-terminus ofthe protein. The loop regions are referred to by the helical regionsthey join, e.g., the A-B loop joins helical bundles A and B. The A-B andC-D loops are long, whereas the B-C loop is short. GH contains fourcysteine residues, all of which participate in disulfide bonds. Thedisulfide assignments are cysteine-53 joined to cysteine-165 andcysteine-182 joined to cysteine-189.

The crystal structure of GH bound to its receptor revealed that GH hastwo receptor binding sites and binds two receptor molecules (Cunninghamet al., 1991; de Vos et al., 1992). The two receptor binding sites arereferred to as site I and site II. Site I encompasses the Carboxy(C)-terminal end of helix D and parts of helix A and the A-B loop,whereas site II encompasses the Amino (N)-terminal region of helix A anda portion of helix C. Binding of GH to its receptor occurs sequentially,with site I always binding first. Site II then engages a second GHreceptor, resulting in receptor dimerization and activation of theintracellular signaling pathways that lead to cellular responses to GH.A GH mutein in which site II has been mutated (a glycine to argininemutation at amino acid 120) is able to bind a single GH receptor, but isunable to dimerize GH receptors; this mutein acts as a GH antagonist invitro, presumably by occupying GH receptor sites without activatingintracellular signaling pathways (Fuh et al., 1992).

The roles of particular regions and amino acids in GH receptor bindingand intracellular signaling also have been studied using techniques suchas mutagenesis, monoclonal antibodies and proteolytic digestion. Thefirst mutagenesis experiments entailed replacing entire domains of GHwith similar regions of the closely related protein, prolactin(Cunningham et al., 1989). One finding was that replacement of the B-Cloop of GH with that of prolactin did not affect binding of the hybridGH protein to a soluble form of the human GH receptor, implying that theB-C loop was non-essential for receptor binding. Alanine scanningmutagenesis (replacement of individual amino acids with alanine)identified 14 amino acids that are critical for GH bioactivity(Cunningham and Wells, 1989). These amino acids are located in thehelices A, B, C, and D and the A-B loop and correspond to sites I and IIidentified from the structural studies. Two lysine residues at aminoacid positions 41 and 172, K41 and K172, were determined to be criticalcomponents of the site I receptor binding site, which explains thedecrease in bioactivity observed when K172 is acetylated (Teh andChapman, 1988). Modification of K168 also significantly reduced GHreceptor binding and bioactivity (de la Llosa et al., 1985; Martal etal., 1985; Teh and Chapman, 1988). Regions of GH responsible for bindingthe GH receptor have also been studied using monoclonal antibodies(Cunningham et al., 1989). A series of eight monoclonal antibodies wasgenerated to human GH and analyzed for the ability to neutralize GHactivity and prevent binding of GH to its recombinant soluble receptor.The latter studies allowed the putative binding site for each monoclonalantibody to be localized within the GH three-dimensional structure. Ofinterest was that monoclonal antibodies 1 and 8 were unable to displaceGH from binding its receptor. The binding sites for these monoclonalantibodies were localized to the B-C loop (monoclonal number 1) and theN-terminal end of the A-B loop (monoclonal number 8). No monoclonalswere studied that bound the C-D loop specifically. The monoclonalantibody studies suggest that the B-C loop and N-terminal end of the A-Bloop are non-essential for receptor binding. Finally, limited cleavageof GH with trypsin was found to produce a two chain derivative thatretained full activity (Mills et al., 1980; Li, 1982). Mapping studiesindicated that trypsin cleaved and/or deleted amino acids betweenpositions 134 and 149, which corresponds to the C-D loop. These studiessuggest the C-D loop is not involved in receptor binding or GHbioactivity.

Structures of a number of cytokines, including G-CSF (Hill et al.,1993), GM-CSF (Diederichs et al., 1991; Walter et al., 1992), IL-2(Bazan, 1992; McKay, 1992), IL-4 (Redfield et al., 1991; Powers et al.,1992), and IL-5 (Milburn et al., 1993) have been determined by X-raydiffraction and NMR studies and show striking conservation with the GHstructure, despite a lack of significant primary sequence homology. EPOis considered to be a member of this family based upon modeling andmutagenesis studies (Boissel et al., 1993; Wen et al., 1994). A largenumber of additional cytokines and growth factors including ciliaryneurotrophic factor (CNTF), leukemia inhibitory factor (LIF),thrombopoietin (TPO), oncostatin M, macrophage colony stimulating factor(M-CSF), IL-3, IL-6, IL-7, IL-9, IL-12, IL-13, IL-15, and alpha, beta,omega, tau and gamma interferon belong to this family (reviewed in Mottand Campbell, 1995; Silvennoinen and Ihle 1996). All of the abovecytokines and growth factors are now considered to comprise one largegene family, of which GH is the prototype.

In addition to sharing similar secondary and tertiary structures,members of this family share the property that they must oligomerizecell surface receptors to activate intracellular signaling pathways.Some GH family members, e.g., GH and EPO, bind a single type of receptorand cause it to form homodimers. Other family members, e.g., IL-2, IL-4,and IL-6, bind more than one type of receptor and cause the receptors toform heterodimers or higher order aggregates (Davis et al., 1993;Paonessa et al., 1995; Mott and Campbell, 1995). Mutagenesis studieshave shown that, like GH, these other cytokines and growth factorscontain multiple receptor binding sites, typically two, and bind theircognate receptors sequentially (Mott and Campbell, 1995; Matthews etal., 1996). Like GH, the primary receptor binding sites for these otherfamily members occur primarily in the four alpha helices and the A-Bloop (reviewed in Mott and Campbell, 1995). The specific amino acids inthe helical bundles that participate in receptor binding differ amongstthe family members (Mott and Campbell, 1995). Most of the cell surfacereceptors that interact with members of the GH supergene family arestructurally related and comprise a second large multi-gene family(Bazan, 1990; Mott and Campbell, 1995; Silvennoinen and Ihle 1996).

A general conclusion reached from mutational studies of various membersof the GH supergene family is that the loops joining the alpha helicesgenerally tend to not be involved in receptor binding. In particular theshort B-C loop appears to be non-essential for receptor binding in most,if not all, family members. For this reason, the B-C loop is a preferredregion for introducing cysteine substitutions in members of the GHsupergene family. The A-B loop, the B-C loop, the C-D loop (and D-E loopof interferon/IL-10-like members of the GH superfamily) also arepreferred sites for introducing cysteine mutations. Amino acids proximalto helix A and distal to the final helix also tend not to be involved inreceptor binding and also are preferred sites for introducing cysteinesubstitutions. Certain members of the GH family, e.g., EPO, IL-2, IL-3,IL-4, IL-6, G-CSF, GM-CSF, TPO, IL-10, IL-12 p35, IL-13, IL-15 andbeta-interferon contain N-linked and O-linked sugars. The glycosylationsites in the proteins occur almost exclusively in the loop regions andnot in the alpha helical bundles. Because the loop regions generally arenot involved in receptor binding and because they are sites for thecovalent attachment of sugar groups, they are preferred sites forintroducing cysteine substitutions into the proteins Amino acids thatcomprise the N- and O-linked glycosylation sites in the proteins arepreferred sites for cysteine substitutions because these amino acids aresurface-exposed, the natural protein can tolerate bulky sugar groupsattached to the proteins at these sites and the glycosylation sites tendto be located away from the receptor binding sites.

Many additional members of the GH gene family are likely to bediscovered in the future. New members of the GH supergene family can beidentified through computer-aided secondary and tertiary structureanalyses of the predicted protein sequences. Members of the GH supergenefamily will possess four or five amphipathic helices joined bynon-helical amino acids (the loop regions). The proteins may contain ahydrophobic signal sequence at their N-terminus to promote secretionfrom the cell. Such later discovered members of the GH supergene familyalso are included within this invention.

The present invention provides “rules” for creating biologically activecysteine-added variants of members of the GH supergene family. These“rules” can be applied to any existing or future member of the GHsupergene family. The cysteine-added variants will posses novelproperties not shared by the naturally occurring proteins. Mostimportantly, the cysteine added variants will possess the property thatthey can be covalently modified with cysteine-reactive polymers or othertypes of cysteine-reactive moieties to generate biologically activeproteins with improved properties such as increased in vivo half-life,increased solubility and improved in vivo efficacy.

Specifically, the present invention provides biologically activecysteine variants of members of the GH supergene family by substitutingcysteine residues for non-essential amino acids in the proteins.Preferably, the cysteine residues are substituted for amino acids thatcomprise the loop regions, for amino acids near the ends of the alphahelices and for amino acids proximal to (preceding) the firstamphipathic helix or distal to (following) the final amphipathic helixof these proteins. Other preferred sites for adding cysteine residuesare at the N-terminus or C-terminus of the proteins. Cysteine residuesalso can be introduced between two amino acids in the disclosed regionsof the polypeptide chain. The present invention teaches that N- andO-linked glycosylation sites in the proteins are preferred sites forintroducing cysteine substitutions either by substitution for aminoacids that make up the sites or, in the case of N-linked sites,introduction of cysteines therein. The glycosylation sites can be serineor threonine residues that are O-glycosylated or asparagine residuesthat are N-glycosylated. N-linked glycosylation sites have the generalstructure asparagine-X-serine or threonine (N-X-S/T), where X can be anyamino acid. The asparagine residue, the amino acid in the X position andthe serine/threonine residue of the N-linked glycosylation site arepreferred sites for creating biologically active cysteine-added variantsof these proteins. Amino acids immediately surrounding or adjacent tothe O-linked and N-linked glycosylation sites (within about 10 residueson either side of the glycosylation site) are preferred sites forintroducing cysteine-substitutions.

More generally, certain of the “rules” for identifying preferred sitesfor creating biologically active cysteine-added protein variants can beapplied to any protein, not just proteins that are members of the GHsupergene family. Specifically, preferred sites for creatingbiologically active cysteine variants of proteins (other than IL-2) areO-linked glycosylation sites. Amino acids immediately surrounding theO-linked glycosylation site (within about 10 residues on either side ofthe glycosylation site) also are preferred sites. N-linked glycosylationsites, and the amino acid residues immediately adjacent on either sideof the glycosylation site (within about 10 residues of the N-X-S/T site)also are preferred sites for creating cysteine added protein variants.Amino acids that can be replaced with cysteine without significant lossof biological activity also are preferred sites for creatingcysteine-added protein variants. Such non-essential amino acids can beidentified by performing cysteine-scanning mutagenesis on the targetprotein and measuring effects on biological activity. Cysteine-scanningmutagenesis entails adding or substituting cysteine residues forindividual amino acids in the polypeptide chain and determining theeffect of the cysteine substitution on biological activity. Cysteinescanning mutagenesis is similar to alanine-scanning mutagenesis(Cunningham et al., 1992), except that target amino acids areindividually replaced with cysteine rather than alanine residues.

Application of the “rules” to create cysteine-added variants andconjugates of protein antagonists also is contemplated. Excessproduction of cytokines and growth factors has been implicated in thepathology of many inflammatory conditions such as rheumatoid arthritis,asthma, allergies and wound scarring. Excess production of GH has beenimplicated as a cause of acromegaly. Certain growth factors andcytokines, e.g., GH and IL-6, have been implicated in proliferation ofparticular cancers. Many of the growth factors and cytokines implicatedin inflammation and cancer are members of the GH supergene family. Thereis considerable interest in developing protein antagonists of thesemolecules to treat these diseases. One strategy involves engineering thecytokines and growth factors so that they can bind to, but notoligomerize receptors. This is accomplished by mutagenizing the secondreceptor binding site (site II) on the molecules. The resulting muteinsare able to bind and occupy receptor sites but are incapable ofactivating intracellular signaling pathways. This strategy has beensuccessfully applied to GH to make a GH antagonist (Cunningham et al.,1992). Similar strategies are being pursued to develop antagonists ofother members of the GH supergene family such as IL-2 (Zurawski et al.,1990; Zurawski and Zurawski, 1992), IL-4 (Kruse et al., 1992), IL-5(Tavernier et al., 1995), GM-CSF (Hercus et al., 1994) and EPO (Matthewset al., 1996). Since the preferred sites for adding cysteine residues tomembers of the GH supergene family described here lie outside of thereceptor binding sites in these proteins, and thus removed from anysites used to create protein antagonists, the cysteine-added variantsdescribed herein could be used to generate long-acting versions ofprotein antagonists. As an example, Cunningham et al. (1992) developedan in vitro GH antagonist by mutating a glycine residue (amino acid 120)to an arginine. This glycine residue is a critical component of thesecond receptor binding site in GH; when it is replaced with arginine,GH cannot dimerize receptors. The glycine to arginine mutation atposition 120 can be introduced into DNA sequences encoding thecysteine-added variants of GH contemplated herein to create acysteine-added GH antagonist that can be conjugated withcysteine-reactive PEGs or other types of cysteine-reactive moieties.Similarly, amino acid changes in other proteins that turn the proteinsfrom agonists to antagonists could be incorporated into DNA sequencesencoding cysteine-added protein variants described herein. Considerableeffort is being spent to identify amino acid changes that convertprotein agonists to antagonists. Hercus et al. (1994) reported thatsubstituting arginine or lysine for glutamic acid at position 21 in themature GM-CSF protein converts GM-CSF from an agonist to an antagonist.Tavernier et al. (1995) reported that substituting glutamine forglutamic acid at position 13 of mature IL-5 creates an IL-5 antagonist.

Experimental strategies similar to those described above can be used tocreate cysteine-added variants (both agonists and antagonists) ofmembers of the GH supergene family derived from various animals. This ispossible because the primary amino acid sequences and structures ofcytokines and growth factors are largely conserved between human andanimal species. For this reason, the “rules” disclosed herein forcreating biologically active cysteine-added variants of members of theGH supergene family will be useful for creating biologically activecysteine-added variants of members of the GH supergene family ofcompanion animals (e.g., dogs, cats, horses) and commercial animal(e.g., cow, sheep, pig) species. Conjugation of these cysteine-addedvariants with cysteine-reactive PEGs will create long-acting versions ofthese proteins that will benefit the companion animal and commercialfarm animal markets. Proteins that are members of the GH supergenefamily (hematopoietic cytokines) are provided in Silvennoimem and Ihle(1996). Silvennoimem and Ihle (1996) also provide information about thestructure and expression of these proteins. DNA sequences, encoded aminoacids and in vitro and in vivo bioassays for the proteins describedherein are described in Aggarwal and Gutterman (1992; 1996), Aggarwal(1998), and Silvennoimem and Ihle (1996). Bioassays for the proteinsalso are provided in catalogues of various commercial suppliers of theseproteins such as R&D Systems, Inc. and Endogen, Inc.

The cysteine variants of the present invention can be used for any ofthe known therapeutic uses of the native proteins in essentially thesame forms and doses all well known in the art. By way of example,therapeutic methods for stimulating platelet production in a patient andfor accelerating recovery from and/or reducing the severity ofthrombocytopenia in a patient are described herein, which use cysteinevariants of interleukin-11 (IL-11) according to the present invention.It is to be understood, however, that general discussion regarding modesof administration, dosage and delivery of cysteine variants such as theIL-11 muteins, is generally intended to apply to therapeutic methodsusing any of the cysteine variants or other PEGylated orcysteine-modified IL-11 proteins described herein.

One embodiment of the present invention relates to cysteine muteins ofIL-11, and methods of making and using such muteins (also referred toherein as IL-11 cysteine variants). IL-11 is a pleiotropic cytokine thatstimulates hematopoiesis, lymphopoeisis and acute phase responses. Theamino acid sequence of human IL-11 (represented herein by SEQ ID NO:17)is given in Kawashima et al. (1991) and Paul et al. (1990) bothincorporated herein by reference. IL-11 is synthesized as a precursorprotein of 199 amino acids that is cleaved to yield a mature protein of178 amino acids. There are no N-linked glycosylation sites in theprotein. IL-11 has four major alpha helices referred to as helices A-D.Relative to the amino acid sequence shown in SEQ ID NO:17, helix Aencompasses amino acids 37-56, helix B encompasses amino acids 92-112,helix C encompasses amino acids 125-147 and helix D encompasses aminoacids 173-196. Amino acids 1-21 of SEQ ID NO:17 encompass the IL-11signal sequence.

This invention provides cysteine-added variants of IL-11, whereincysteine substitutions or insertions are made in one or more of theregion proximal to (preceding) the A helix (amino acids 22-36 of SEQ IDNO:17), distal to (following) the D helix (amino acids 197-199 of SEQ IDNO:17), in the A-B loop (amino acids 57-91 of SEQ ID NO:17), in the B-Cloop (amino acids 113-124 of SEQ ID NO:17), and in the C-D loop (aminoacids 148-172 of SEQ ID NO:17). This invention also providescysteine-added variants at the first three or last three amino acids inany one or more of helices A, B, C and D. Variants in which cysteineresidues are added proximal to the first amino acid of the matureprotein (amino acid 22 of SEQ ID NO:17) or distal to the final aminoacid of the mature protein (amino acid 199 of SEQ ID NO:17) also areprovided. Any individual amino acid encompassed by the above-identifieddisclosure of regions is expressly included as a residue for cysteinesubstitution, or before or after which a cysteine can be inserted,according to the present invention.

Preferred site for cysteine substitutions in the region preceding helixA of IL-11 are (relative to SEQ ID NO:17): P22, G23, P24, P25, P26, G27,P28, P29, R30, V31, S32, P33, D34, P35, and R36. Preferred sites forcysteine substitutions in the A-B loop of IL-11 are (relative to SEQ IDNO:17): A57, A58, Q59, L60, R61, D62, K63, F64, P65, A66, D67, G68, D69,H70, N71, L72, D73, S74, L75, P76, T77, L78, A79, M80, S81, A82, G83,A84, L85, G86, A87, L88, Q89, L90, and P91. Preferred sites for cysteinesubstitutions in the C-D loop of IL-11 are (relative to SEQ ID NO:17):A148, L149, P150, Q151, P152, P153, P154, D155, P156, P157, A158, P159,P160, L161, A162, P163, P164, S165, 5166, A167, W168, G169, G170, I171,and R172. Preferred sites for cysteine substitutions in region followinghelix D of IL-11 are (relative to SEQ ID NO:17): T197, R198, and L199.Preferred sites for cysteine substitutions in the first 3 amino acids ofhelix A are (relative to SEQ ID NO:17): A37, D38, and L39. Preferredsites for cysteine substitutions in the last 3 amino acids of helix Aare (relative to SEQ ID NO:17): R54, Q55, and L56. Preferred sites forcysteine substitutions in the first 3 amino acids of helix B are(relative to SEQ ID NO:17): G92, V93, and L94. Preferred sites forcysteine substitutions in the last 3 amino acids of helix B are(relative to SEQ ID NO:17): W110, L111, and R112. Preferred sites forcysteine substitutions in the first 3 amino acids of helix C are(relative to SEQ ID NO:17): E125, L126, and G127. Preferred sites forcysteine substitutions in the last 3 amino acids of helix C are(relative to SEQ ID NO:17): 5145, R146, and L147. Preferred sites forcysteine substitutions in the first 3 amino acids of helix D are(relative to SEQ ID NO:17): A173, A174, and H175. Preferred sites forcysteine substitutions in the last 3 amino acids of helix D are(relative to SEQ ID NO:17): L194, L195, and K196.

As used herein, reference to an isolated protein or polypeptide in thepresent invention, including an IL-11 protein described particularlyherein, includes full-length proteins, fusion proteins, or any fragment(truncated form) or homologue of such a protein. Such a protein caninclude, but is not limited to, purified proteins, recombinantlyproduced proteins, membrane bound proteins, proteins complexed withlipids, soluble proteins and isolated proteins associated with otherproteins. More specifically, an isolated protein according to thepresent invention, is a protein (including a polypeptide or peptide)that has been removed from its natural milieu (i.e., that has beensubject to human manipulation) and can include purified proteins,partially purified proteins, recombinantly produced proteins, andsynthetically produced proteins, for example. As such, “isolated” doesnot reflect the extent to which the protein has been purified.Preferably, an isolated protein of the present invention is producedrecombinantly. In addition, and again by way of example, a “human IL-11protein” or a protein “derived from” a human IL-11 protein refers to aIL-11 protein (generally including a homologue of a naturally occurringIL-11 protein) from a human (Homo sapiens) or to a IL-11 protein thathas been otherwise produced from the knowledge of the structure (e.g.,sequence) and perhaps the function of a naturally occurring IL-11protein from Homo sapiens. In other words, a human IL-11 proteinincludes any IL-11 protein that has substantially similar structure andfunction of a naturally occurring IL-11 protein from Homo sapiens orthat is a biologically active (i.e., has biological activity) homologueof a naturally occurring IL-11 protein from Homo sapiens as described indetail herein. As such, a human IL-11 protein can include purified,partially purified, recombinant, mutated/modified and syntheticproteins. According to the present invention, the terms “modification”and “mutation” can be used interchangeably, particularly with regard tothe modifications/mutations to the amino acid sequence of protein (ornucleic acid sequences) described herein. An isolated protein useful asan antagonist or agonist according to the present invention can beisolated from its natural source, produced recombinantly or producedsynthetically.

As used herein, the term “homologue” is used to refer to a protein orpeptide which differs from a naturally occurring protein or peptide(i.e., the “prototype” or “wild-type” protein) by modifications,including minor modifications, to the naturally occurring protein orpeptide, but which maintains the basic protein and side chain structureof the naturally occurring form. Such changes include, but are notlimited to: changes in one or a few (i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9 or10) amino acid side chains; changes one or a few amino acids, includingdeletions (e.g., a truncated form of the protein or peptide) insertionsand/or substitutions; changes in stereochemistry of one or a few atoms;and/or minor derivatizations, including but not limited to: methylation,glycosylation, phosphorylation, acetylation, myristoylation,prenylation, palmitation, amidation and/or addition ofglycosylphosphatidyl inositol. A homologue can have either enhanced,decreased, or substantially similar properties as compared to thenaturally occurring protein or peptide. A homologue can include anagonist of a protein or an antagonist of a protein. A cysteine variantof IL-11 is a homologue of IL-11.

Homologues can be the result of natural allelic variation or naturalmutation. A naturally occurring allelic variant of a nucleic acidencoding a protein is a gene that occurs at essentially the same locus(or loci) in the genome as the gene which encodes such protein, butwhich, due to natural variations caused by, for example, mutation orrecombination, has a similar but not identical sequence. Allelicvariants typically encode proteins having similar activity to that ofthe protein encoded by the gene to which they are being compared. Oneclass of allelic variants can encode the same protein but have differentnucleic acid sequences due to the degeneracy of the genetic code.Allelic variants can also comprise alterations in the 5′ or 3′untranslated regions of the gene (e.g., in regulatory control regions).Allelic variants are well known to those skilled in the art.

Homologues can be produced using techniques known in the art for theproduction of proteins including, but not limited to, directmodifications to the isolated, naturally occurring protein, directprotein synthesis, or modifications to the nucleic acid sequenceencoding the protein using, for example, classic or recombinant DNAtechniques to effect random or targeted mutagenesis.

In one embodiment, a homologue of a given protein comprises, consistsessentially of, or consists of, an amino acid sequence that is at leastabout 45%, or at least about 50%, or at least about 55%, or at leastabout 60%, or at least about 65%, or at least about 70%, or at leastabout 75%, or at least about 80%, or at least about 85%, or at leastabout 90%, or at least about 95% identical, or at least about 95%identical, or at least about 96% identical, or at least about 97%identical, or at least about 98% identical, or at least about 99%identical (or any percent identity between 45% and 99%, in whole integerincrements), to the amino acid sequence of the reference protein. In oneembodiment, the homologue comprises, consists essentially of, orconsists of, an amino acid sequence that is less than 100% identical,less than about 99% identical, less than about 98% identical, less thanabout 97% identical, less than about 96% identical, less than about 95%identical, and so on, in increments of 1%, to less than about 70%identical to the naturally occurring amino acid sequence of thereference protein.

As used herein, unless otherwise specified, reference to a percent (%)identity refers to an evaluation of homology which is performed using:(1) a BLAST 2.0 Basic BLAST homology search using blastp for amino acidsearches and blastn for nucleic acid searches with standard defaultparameters, wherein the query sequence is filtered for low complexityregions by default (described in Altschul, S. F., Madden, T. L.,Schääffer, A. A., Zhang, J., Zhang, Z., Miller, W. & Lipman, D. J.(1997) “Gapped BLAST and PSI-BLAST: a new generation of protein databasesearch programs.” Nucleic Acids Res. 25:3389-3402, incorporated hereinby reference in its entirety); (2) a BLAST 2 alignment (using theparameters described below); (3) and/or PSI-BLAST with the standarddefault parameters (Position-Specific Iterated BLAST. It is noted thatdue to some differences in the standard parameters between BLAST 2.0Basic BLAST and BLAST 2, two specific sequences might be recognized ashaving significant homology using the BLAST 2 program, whereas a searchperformed in BLAST 2.0 Basic BLAST using one of the sequences as thequery sequence may not identify the second sequence in the top matches.In addition, PSI-BLAST provides an automated, easy-to-use version of a“profile” search, which is a sensitive way to look for sequencehomologues. The program first performs a gapped BLAST database search.The PSI-BLAST program uses the information from any significantalignments returned to construct a position-specific score matrix, whichreplaces the query sequence for the next round of database searching.Therefore, it is to be understood that percent identity can bedetermined by using any one of these programs.

Two specific sequences can be aligned to one another using BLAST 2sequence as described in Tatusova and Madden, (1999), “Blast 2sequences—a new tool for comparing protein and nucleotide sequences”,FEMS Microbiol Lett. 174:247-250, incorporated herein by reference inits entirety. BLAST 2 sequence alignment is performed in blastp orblastn using the BLAST 2.0 algorithm to perform a Gapped BLAST search(BLAST 2.0) between the two sequences allowing for the introduction ofgaps (deletions and insertions) in the resulting alignment. For purposesof clarity herein, a BLAST 2 sequence alignment is performed using thestandard default parameters as follows.

For blastn, using 0 BLOSUM62 matrix:

Reward for match=1

Penalty for mismatch=−2

Open gap (5) and extension gap (2) penalties

gap x_dropoff (50) expect (10) word size (11) filter (on)

For blastp, using 0 BLOSUM62 matrix:

Open gap (11) and extension gap (1) penalties

gap x_dropoff (50) expect (10) word size (3) filter (on).

According to the present invention, an isolated IL-11 protein, includinga biologically active homologue or fragment thereof, has at least onecharacteristic of biological activity of activity the wild-type, ornaturally occurring IL-11 protein (which can vary depending on whetherthe homologue or fragment is an agonist or antagonist of the protein, orwhether an agonist or antagonist mimetic of the protein is described).In general, the biological activity or biological action of a proteinrefers to any function(s) exhibited or performed by the protein that isascribed to the naturally occurring form of the protein as measured orobserved in vivo (i.e., in the natural physiological environment of theprotein) or in vitro (i.e., under laboratory conditions). Modifications,activities or interactions which result in a decrease in proteinexpression or a decrease in the activity of the protein, can be referredto as inactivation (complete or partial), down-regulation, reducedaction, or decreased action or activity of a protein. Similarly,modifications, activities or interactions which result in an increase inprotein expression or an increase in the activity of the protein, can bereferred to as amplification, overproduction, activation, enhancement,up-regulation or increased action of a protein. The biological activityof an IL-11 protein according to the invention can be measured orevaluated using any assay for the biological activity of the protein asknown in the art. Such assays are known in the art, and assays for IL-11activity are described in the Examples.

In accordance with the present invention, an isolated polynucleotide(also referred to as an isolated nucleic acid molecule) is a nucleicacid molecule that has been removed from its natural milieu (e.g., thathas been subject to human manipulation), its natural milieu being thegenome or chromosome in which the nucleic acid molecule is found innature. As such, “isolated” does not necessarily reflect the extent towhich the nucleic acid molecule has been purified, but indicates thatthe molecule does not include an entire genome or an entire chromosomein which the nucleic acid molecule is found in nature. A polynucleotideuseful in the present invention can include a portion of a nucleic acidsequence (sense or non-sense strand) that is suitable for use as ahybridization probe or PCR primer for the identification of afull-length gene (or portion thereof), or to encode a protein orfragment (truncated form) or homologue thereof. An isolated nucleic acidmolecule that includes a gene is not a fragment of a chromosome thatincludes such gene, but rather includes the coding region and regulatoryregions associated with the gene, but no additional genes naturallyfound on the same chromosome. An isolated nucleic acid molecule can alsoinclude a specified nucleic acid sequence flanked by (i.e., at the 5′and/or the 3′ end of the sequence) additional nucleic acids that do notnormally flank the specified nucleic acid sequence in nature (i.e.,heterologous sequences). Isolated nucleic acid molecule can include DNA,RNA (e.g., mRNA), or derivatives of either DNA or RNA (e.g., cDNA).Although the phrase “nucleic acid molecule” primarily refers to thephysical nucleic acid molecule and the phrase “nucleic acid sequence”primarily refers to the sequence of nucleotides on the nucleic acidmolecule, the two phrases can be used interchangeably, especially withrespect to a nucleic acid molecule, or a nucleic acid sequence, beingcapable of encoding a protein. Preferably, an isolated nucleic acidmolecule of the present invention is produced using recombinant DNAtechnology (e.g., polymerase chain reaction (PCR) amplification,cloning) or chemical synthesis.

The minimum size of a nucleic acid molecule or polynucleotide of thepresent invention is a size sufficient to encode a protein having adesired biological activity, or sufficient to form a probe oroligonucleotide primer that is capable of forming a stable hybrid withthe complementary sequence of a nucleic acid molecule encoding thenatural protein (e.g., under moderate, high or very high stringencyconditions). If the polynucleotide is an oligonucleotide probe orprimer, the size of the polynucleotide can be dependent on nucleic acidcomposition and percent homology or identity between the nucleic acidmolecule and a complementary sequence as well as upon hybridizationconditions per se (e.g., temperature, salt concentration, and formamideconcentration). The minimum size of a polynucleotide that is used as anoligonucleotide probe or primer is at least about 5 nucleotides inlength, and preferably ranges from about 5 to about 50 or about 500nucleotides or greater, including any length in between, in whole numberincrements (i.e., 5, 6, 7, 8, 9, 10, . . . 33, 34, . . . 256, 257, . . .500). There is no limit, other than a practical limit, on the maximalsize of a nucleic acid molecule of the present invention, in that thenucleic acid molecule can include a portion of a protein-encodingsequence or a nucleic acid sequence encoding a full-length protein.

As used herein, stringent hybridization conditions refer to standardhybridization conditions under which nucleic acid molecules are used toidentify similar nucleic acid molecules. Such standard conditions aredisclosed, for example, in Sambrook et al., Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Labs Press, 1989. Sambrook et al.,ibid., is incorporated by reference herein in its entirety (seespecifically, pages 9.31-9.62). In addition, formulae to calculate theappropriate hybridization and wash conditions to achieve hybridizationpermitting varying degrees of mismatch of nucleotides are disclosed, forexample, in Meinkoth et al., 1984, Anal. Biochem. 138, 267-284; Meinkothet al., ibid., is incorporated by reference herein in its entirety.

More particularly, moderate stringency hybridization and washingconditions, as referred to herein, refer to conditions which permitisolation of nucleic acid molecules having at least about 70% nucleicacid sequence identity with the nucleic acid molecule being used toprobe in the hybridization reaction (i.e., conditions permitting about30% or less mismatch of nucleotides). High stringency hybridization andwashing conditions, as referred to herein, refer to conditions whichpermit isolation of nucleic acid molecules having at least about 80%nucleic acid sequence identity with the nucleic acid molecule being usedto probe in the hybridization reaction (i.e., conditions permittingabout 20% or less mismatch of nucleotides). Very high stringencyhybridization and washing conditions, as referred to herein, refer toconditions which permit isolation of nucleic acid molecules having atleast about 90% nucleic acid sequence identity with the nucleic acidmolecule being used to probe in the hybridization reaction (i.e.,conditions permitting about 10% or less mismatch of nucleotides). Asdiscussed above, one of skill in the art can use the formulae inMeinkoth et al., ibid. to calculate the appropriate hybridization andwash conditions to achieve these particular levels of nucleotidemismatch. Such conditions will vary, depending on whether DNA:RNA orDNA:DNA hybrids are being formed. Calculated melting temperatures forDNA:DNA hybrids are 10° C. less than for DNA:RNA hybrids. In particularembodiments, stringent hybridization conditions for DNA:DNA hybridsinclude hybridization at an ionic strength of 6×SSC (0.9 M Na⁺) at atemperature of between about 20° C. and about 35° C. (lower stringency),more preferably, between about 28° C. and about 40° C. (more stringent),and even more preferably, between about 35° C. and about 45° C. (evenmore stringent), with appropriate wash conditions. In particularembodiments, stringent hybridization conditions for DNA:RNA hybridsinclude hybridization at an ionic strength of 6×SSC (0.9 M Na⁺) at atemperature of between about 30° C. and about 45° C., more preferably,between about 38° C. and about 50° C., and even more preferably, betweenabout 45° C. and about 55° C., with similarly stringent wash conditions.These values are based on calculations of a melting temperature formolecules larger than about 100 nucleotides, 0% formamide and a G+Ccontent of about 40%. Alternatively, T_(m) can be calculated empiricallyas set forth in Sambrook et al., supra, pages 9.31 to 9.62. In general,the wash conditions should be as stringent as possible, and should beappropriate for the chosen hybridization conditions. For example,hybridization conditions can include a combination of salt andtemperature conditions that are approximately 20-25° C. below thecalculated T_(m) of a particular hybrid, and wash conditions typicallyinclude a combination of salt and temperature conditions that areapproximately 12-20° C. below the calculated T_(m) of the particularhybrid. One example of hybridization conditions suitable for use withDNA:DNA hybrids includes a 2-24 hour hybridization in 6×SSC (50%formamide) at about 42° C., followed by washing steps that include oneor more washes at room temperature in about 2×SSC, followed byadditional washes at higher temperatures and lower ionic strength (e.g.,at least one wash as about 37° C. in about 0.1×-0.5×SSC, followed by atleast one wash at about 68° C. in about 0.1×-0.5×SSC).

In one embodiment of the present invention, any of the amino acidsequences described herein, including homologues of such sequences(e.g., cysteine muteins), can be produced with from at least one, and upto about 20, additional heterologous amino acids flanking each of the C-and/or N-terminal end of the given amino acid sequence. The resultingprotein or polypeptide can be referred to as “consisting essentially of”a given amino acid sequence. According to the present invention, theheterologous amino acids are a sequence of amino acids that are notnaturally found (i.e., not found in nature, in vivo) flanking the givenamino acid sequence or which would not be encoded by the nucleotidesthat flank the naturally occurring nucleic acid sequence encoding thegiven amino acid sequence as it occurs in the gene, if such nucleotidesin the naturally occurring sequence were translated using standard codonusage for the organism from which the given amino acid sequence isderived. Similarly, the phrase “consisting essentially of”, when usedwith reference to a nucleic acid sequence herein, refers to a nucleicacid sequence encoding a given amino acid sequence that can be flankedby from at least one, and up to as many as about 60, additionalheterologous nucleotides at each of the 5′ and/or the 3′ end of thenucleic acid sequence encoding the given amino acid sequence. Theheterologous nucleotides are not naturally found (i.e., not found innature, in vivo) flanking the nucleic acid sequence encoding the givenamino acid sequence as it occurs in the natural gene.

One embodiment of the present invention relates to a method to produce acysteine mutein of IL-11, including any cystein mutein of IL-11described herein. Such a method includes the steps of production andpurification of the IL-11 mutein using an insect system as described indetail any of Examples 2-4, using an E. coli system as described indetail in Examples 7-8, or using an E. coli intein system as describedin detail Example 9.

One embodiment of the present invention relates to stimulatingproduction of platelets in an animal, comprising administering to theanimal an interleukin-11 (IL-11) cysteine mutein as described herein, ora composition comprising such a cysteine mutein, and in one embodiment,as prepared using methods described herein (see Examples) oralternatively, as described in PCT Publication No. WO 01/87925,published Nov. 22, 2001, or in PCT Publication No. WO 00/42175,published Jul. 20, 2000, each of which is incorporated herein byreference in its entirety.

One embodiment of the invention relates to a method to treat or protectan animal from a disease or condition that is amenable to treatment withinterleukin-11 (IL-11), comprising administering to the animal acomposition comprising an IL-11 cysteine mutein as described herein. Inone embodiment, the cysteine mutein is prepared using methods describedherein or in PCT Publication No. WO 01/87925 or in PCT Publication No.WO 00/42175, supra.

Another embodiment of the invention relates to a method to prevent ortreat the occurrence of thrombocytopenia in an animal comprisingadministering to the animal a composition comprising an IL-11 cysteinemutein as described herein and in one embodiment, as prepared usingmethods described herein or in PCT Publication No. WO 01/87925 or in PCTPublication No. WO 00/42175, supra. In a preferred aspect of theinvention, administration of the IL-11 cysteine mutein acceleratesrecovery from thrombocytopenia and/or reduces the severity ofthrombocytopenia in the patient. The thrombocytopenia to be prevented ortreated using this method can include, but is not limited to: (a)thrombocytopenia resulting from myelosuppressive chemotherapy; (b)thrombocytopenia resulting from other chemical treatments; (c)thrombocytopenia resulting from radiological treatments; (d)thrombocytopenia resulting from disease; (e) thrombocytopenia resultingfrom idiopathic causes; (f) thrombocytopenia resulting from drugtreatments, including interferons and ribavarin; (g) thrombocytopenia inneonates; (h) thrombocytopenia resulting from myelodysplastic syndromes;(i) thrombocytopenia resulting from aplastic anemia; and (j)thrombocytopenia resulting from cirrhosis.

Approximately 1.4 million people receive myelosuppressive chemotherapyeach year in the U.S. Thrombocytopenia is one of the most commonhematological complications of chemotherapy (occurs in 40-60% ofpatients) and can lead to dose reductions or delays in chemotherapy,which reduce effectiveness of the chemotherapy treatment and adverselyaffect patient survival (reviewed in Cairo, 2000). Thrombocytopenia is acommon problem in other disease indications besides cancer. For example,thrombocytopenia is the most common hematological abnormality seen inneonates (reviewed in Ramasethu, 2004). Thrombocytopenia is a majorcomplication of PEGylated interferon/ribavarin treatments in patientswith Hepatitis C, and can lead to dose-reductions or delays inanti-viral therapy, which can adversely affect treatment outcome(Dieterich and Spivak, 2003). A subset of patients with bone marrowdisorders such as myelodysplastic syndromes and aplastic anemia respondto IL-11 therapy by reversing their thrombocytopenia (Gordon, 1999;Kurzrock et al., 2001; Tsimberidou et al., 2005). IL-11 also iseffective at reversing thrombocytopenia in patients with cirrhosis(Ghalib et al., 2003). Thus, there are many potential clinical settingswhere a composition comprising an IL-11 cysteine mutein as describedherein will prove useful.

IL-11, like many cytokines, has a short circulating half-life, whichnecessitates daily subcutaneous injections for maximum effectiveness inhumans. The present inventors have created novel IL-11 analogs (e.g.,the IL-11 cysteine muteins described herein) with improved in vivocharacteristics such as increased circulating half-life and improvedtherapeutic efficacy through site-specific chemical modification of theprotein with Polyethylene Glycol (PEG) reagents (e.g., usingcysteine-reactive PEGylation and/or by PEGylation of other residues inthe protein). Many of these analogs were created by introducing a “free”cysteine residue (i.e., a cysteine residue not involved in a disulfidebond) into the protein using site-directed mutagenesis. The freecysteine residue(s) serve as the site for covalent modification of theprotein with cysteine-reactive PEG reagents. The present inventionteaches a variety of human IL-11 cysteine muteins that can be modifiedwith cysteine-reactive PEG reagents and retain biological activity, andthe Examples section below describes additional methods of preparingsuch muteins.

Human and rodent IL-11 proteins perform similar functions in theirrespective species and studies with rodent IL-11 proteins can be used topredict the function of human IL-11 in humans. Human and rodent IL-11proteins share about 69% amino acid identity, and have cross-speciescross-reactivity in terms of biological activity and receptor binding.Indeed, the Examples below demonstrate the use of a human IL-11 cysteinemutein in in vivo studies using rats. It is possible to use the aminoacid identity between human and rodent IL-11 proteins to construct humanIL-11 cysteine muteins that can be expressed, purified and PEGylatedusing procedures described herein, and the biological activities of thePEGylated IL-11 cysteine muteins can be tested in rodent animal diseasemodels and used to predict the effectiveness of PEGylated human IL-11cysteine variants in humans. Similarly, one can construct rodent analogsof human IL-11 cysteine variants to be used in rodent animal diseasemodels and again predict the effectiveness of corresponding orequivalent PEGylated human cysteine variants.

A preferred embodiment of the present invention is a PEGylated IL-11protein. A more preferred embodiment is a monoPEGylated IL-11 protein.MonoPEGylated indicates that the protein is modified with a single PEG(i.e., at a single site in the protein). It is well known in the artthat PEGylated proteins can have widely varying in vitro bioactivitiesdue to where the PEG attaches to the protein. A preferred composition ofthe present invention is a PEGylated or monoPEGylated IL-11 analog thathas in vitro bioactivity (EC₅₀s) of less than about 1000 ng/mL in anIL-11 dependent in vitro bioassay. The preferred IL-11-dependentbioassay is the IL-11-dependent cell proliferation bioassay using B9-11cells, described herein. A more preferred composition is a PEGylated ormonoPEGylated IL-11 analog with an EC₅₀ of less than about 100 ng/mL inan in vitro bioassay. An even more preferred composition is a PEGylatedor monoPEGylated IL-11 protein with an EC₅₀ less than about 20 ng/mL inan in vitro bioassay. The Examples presented below teach preferredmethods for preparing PEGylated and monoPEGylated IL-11 analogs thathave the in vitro bioactivities described above.

Toward that end, the present inventors have constructed multiple IL-11cysteine muteins, as discussed above, and have and tested the humanIL-11 *200C mutein in in vivo assays for IL-11 activity. As shown in theExamples, the PEGylated human IL-11 cysteine variant is effective atstimulating platelet production in a mammal (a rodent), which indicatesthat PEGylated human IL-11 cysteine muteins will be effective atstimulating platelet production in humans. Stimulating plateletproduction will be useful for ameliorating disease indications in whichsuch activity is impaired, such as thrombocytopenia. Indeed, thePEGylated IL-11 cysteine variant was further shown (see Examples) toaccelerate recovery and reduce the severity of thrombocytopenia in a ratanimal model. In the rat model, the thrombocytopenia was induced bymyelosuppression, but the present invention is useful for treatingthrombocytopenia resulting from any cause (chemical, radiological,disease, etc.). In addition, the Examples demonstrate that the IL-11cysteine variants of the invention have a superior half-life as comparedto the wild-type protein, may have better activity than the wild-typeprotein, and may have in vivo efficacy after a single injection, where asingle injection of wild-type IL-11 had no detectable effect in vivo.The other cysteine variants described in the Examples are similarlyexpected to be useful in in vivo methods according to the invention.

As discussed above, various embodiments of the invention relate tomethods of use of IL-11 cysteine muteins. In particular, the presentinvention relates to the use of these muteins to protect an animal froma disease or condition that is amenable to treatment by the use ofwild-type IL-11, or which might be particularly amenable to treatmentusing the IL-11 cysteine muteins of the present invention.

As used herein, the phrase “protected from a disease” refers to reducingthe symptoms of the disease, reducing the occurrence of the disease,and/or reducing the severity of the disease. Protecting an animal canrefer to the ability of a therapeutic composition of the presentinvention, when administered to an animal, to prevent a disease fromoccurring and/or to cure or to alleviate disease symptoms, signs orcauses. As such, to protect an animal from a disease includes bothpreventing disease occurrence (prophylactic treatment) and treating ananimal that has a disease or that is experiencing initial symptoms of adisease (therapeutic treatment). In particular, protecting an animalfrom a disease is accomplished by inducing a beneficial or protectivetherapeutic response in the animal by administration of an IL-11cysteine mutein, or any of the other cysteine muteins of the presentinvention as described herein. The term, “disease” refers to anydeviation from the normal health of a mammal and includes a state whendisease symptoms are present, as well as conditions in which a deviation(e.g., infection, gene mutation, genetic defect, etc.) has occurred, butsymptoms are not yet manifested.

Accordingly, an IL-11 cysteine mutein of the present invention can beadministered to regulate the stimulation of platelet production in ananimal. An IL-11 cysteine mutein can be administered to an animal toprevent or ameliorate any disease or condition for which the use of thewild-type protein can be used. Such diseases and conditions aredescribed in detail above. In one embodiment, an IL-11 cysteine muteinof the present invention is used to protect or treat an animal that hasor is at risk of developing thrombocytopenia, and specificallyaccelerates the recovery from thrombocytopenia and/or reduces theseverity of the thrombocytopenia in the animal.

An IL-11 cysteine mutein or composition comprising the same of thepresent invention is administered to an animal in a manner effective todeliver the composition to a target cell, a target tissue, orsystemically to the animal, whereby provision of a therapeutic benefitis achieved as a result of the administration of the mutein orcomposition. Suitable administration protocols include any in vivo or exvivo administration protocol. According to the present invention,suitable methods of administering a composition of the present inventionto a patient include any route of in vivo administration that issuitable for delivering the composition into a patient. The preferredroutes of administration will be apparent to those of skill in the art,depending on the type of condition to be prevented or treated and/or thetarget cell population.

Cysteine muteins of the present invention are preferably administered ina composition. Compositions can include a cysteine mutein of theinvention and any other suitable pharmaceutically acceptable carrier, aswell as, in some aspects, additional components that may be useful inthe treatment of a give disease or condition. According to the presentinvention, a “pharmaceutically acceptable carrier” includespharmaceutically acceptable excipients and/or pharmaceuticallyacceptable delivery vehicles, which are suitable for use inadministration of the composition to a suitable in vitro, ex vivo or invivo site. A suitable in vitro, in vivo or ex vivo site is preferablyany site where the cysteine mutein will provide a detectable effect ascompared to in the absence of the mutein, and includes a disease site ora site of cell types to be contacted with the mutein. Preferredpharmaceutically acceptable carriers are capable of maintaining themutein of the present invention in a form that, upon arrival of themutein at the cell target in a culture or in patient, the mutein iscapable of interacting with its target (e.g., platelets or progenitorcells thereof).

Suitable excipients of the present invention include excipients orformularies that transport or help transport, but do not specificallytarget a composition to a cell or area (also referred to herein asnon-targeting carriers). Examples of pharmaceutically acceptableexcipients include, but are not limited to water, phosphate bufferedsaline, Ringer's solution, dextrose solution, serum-containingsolutions, Hank's solution, other aqueous physiologically balancedsolutions, oils, esters and glycols. Aqueous carriers can containsuitable auxiliary substances required to approximate the physiologicalconditions of the recipient, for example, by enhancing chemicalstability and isotonicity. Compositions of the present invention can besterilized by conventional methods and/or lyophilized

One type of pharmaceutically acceptable carrier includes a controlledrelease formulation that is capable of slowly releasing a composition ofthe present invention into a patient or culture. As used herein, acontrolled release formulation comprises a cysteine mutein of thepresent invention in a controlled release vehicle. Suitable controlledrelease vehicles include, but are not limited to, biocompatiblepolymers, other polymeric matrices, capsules, microcapsules,microparticles, bolus preparations, osmotic pumps, diffusion devices,liposomes, lipospheres, and transdermal delivery systems. Other carriersof the present invention include liquids that, upon administration to apatient, form a solid or a gel in situ. Preferred carriers are alsobiodegradable (i.e., bioerodible). In the event that a cysteine muteinof the invention is administered as a recombinant nucleic acid moleculeencoding the cysteine mutein (e.g., gene therapy or geneticimmunization), suitable carriers include, but are not limited toliposomes, viral vectors or other carriers, including ribozymes, goldparticles, poly-L-lysine/DNA-molecular conjugates, and artificialchromosomes. Natural lipid-containing carriers include cells andcellular membranes. Artificial lipid-containing carriers includeliposomes and micelles.

A carrier of the present invention can be modified to target to aparticular site in a patient, thereby targeting and making use of acompound of the present invention at that site. A pharmaceuticallyacceptable carrier which is capable of targeting can also be referred toherein as a “delivery vehicle” or “targeting carrier”. Suitablemodifications include manipulating the chemical formula of the lipidportion of the delivery vehicle and/or introducing into the vehicle atargeting agent capable of specifically targeting a delivery vehicle toa preferred site or target site, for example, a preferred cell type. A“target site” refers to a site in a patient to which one desires todeliver a composition. Suitable targeting compounds include ligandscapable of selectively (i.e., specifically) binding another molecule ata particular site. Examples of such ligands include antibodies,antigens, receptors and receptor ligands. Manipulating the chemicalformula of the lipid portion of the delivery vehicle can modulate theextracellular or intracellular targeting of the delivery vehicle. Forexample, a chemical can be added to the lipid formula of a liposome thatalters the charge of the lipid bilayer of the liposome so that theliposome fuses with particular cells having particular chargecharacteristics.

One delivery vehicle of the present invention is a liposome. A liposomeis capable of remaining stable in an animal for a sufficient amount oftime to deliver a nucleic acid molecule or protein described in thepresent invention to a preferred site in the animal. A liposome,according to the present invention, comprises a lipid composition thatis capable of delivering a nucleic acid molecule or protein to aparticular, or selected, site in a patient. A liposome according to thepresent invention comprises a lipid composition that is capable offusing with the plasma membrane of the targeted cell to deliver anucleic acid molecule or protein into a cell. Suitable liposomes for usewith the present invention include any liposome. Preferred liposomes ofthe present invention include those liposomes commonly used in, forexample, gene delivery methods known to those of skill in the art. Morepreferred liposomes comprise liposomes having a polycationic lipidcomposition and/or liposomes having a cholesterol backbone conjugated topolyethylene glycol. Complexing a liposome with a nucleic acid moleculeor protein of the present invention can be achieved using methodsstandard in the art.

Another type of delivery vehicle, when the cysteine mutein isadministered as a nucleic acid encoding the mutein, comprises a viralvector. A viral vector includes an isolated nucleic acid molecule, inwhich the nucleic acid molecules are packaged in a viral coat thatallows entrance of DNA into a cell. A number of viral vectors can beused, including, but not limited to, those based on alphaviruses,poxviruses, adenoviruses, herpesviruses, lentiviruses, adeno-associatedviruses and retroviruses.

According to the present invention, an effective administration protocol(i.e., administering a therapeutic composition in an effective manner)comprises suitable dose parameters and modes of administration thatresult in the desired effect in the patient (e.g., stimulation ofplatelet production), preferably so that the patient is protected fromthe disease (e.g., by disease prevention or by alleviating one or moresymptoms of ongoing disease). Effective dose parameters can bedetermined using methods standard in the art for a particular disease.Such methods include, for example, determination of survival rates, sideeffects (i.e., toxicity) and progression or regression of disease.

In accordance with the present invention, a suitable single dose size isa dose that results in the desired therapeutic effect in the patient,depending on the cysteine mutein that is administered, or in theamelioration of at least one symptom of a condition in the patient, whenadministered one or more times over a suitable time period. Doses canvary depending upon the disease being treated. One of skill in the artcan readily determine appropriate single dose sizes for a given patientbased on the size of a patient and the route of administration.

In one aspect of the invention, a suitable single dose of a therapeuticcomposition of the present invention is an amount that, whenadministered by any route of administration, provides a therapeuticeffect in the patient as described above, as compared to a patient whichhas not been administered with the therapeutic composition of thepresent invention (i.e., a control patient), as compared to the patientprior to administration of the composition, or as compared to a standardestablished for the particular disease, patient type and composition.

In one aspect of the invention an appropriate single dose of a cysteinemutein of the present invention is at least about 0.01 micrograms per kgof the animal to which the mutein is administered, and in other aspects,at least about 0.1 micrograms/kg, at least about 0.2 micrograms/kg, atleast about 0.5 micrograms/kg, at least about 1 micrograms/kg, at leastabout 5 micrograms/kg, at least about 10 micrograms/kg, at least about25 micrograms/kg, at least about 50 micrograms/kg, at least about 75micrograms/kg, at least about 100 micrograms/kg, at least about 200micrograms/kg, at least about 300 micrograms/kg, at least about 400micrograms/kg, at least about 500 micrograms/kg, at least about 750micrograms/kg, at least about 1 mg/kg, or at least about 5 mg/kg.

As discussed above, a therapeutic composition of the present inventionis administered to a patient in a manner effective to deliver thecomposition to a cell, a tissue, and/or systemically to the patient,whereby the desired result is achieved as a result of the administrationof the composition. Suitable administration protocols include any invivo or ex vivo administration protocol. The preferred routes ofadministration will be apparent to those of skill in the art, dependingon the type of condition to be prevented or treated; whether thecomposition is nucleic acid based or protein based; and/or the targetcell/tissue. For proteins or nucleic acid molecules, preferred methodsof in vivo administration include, but are not limited to, intravenousadministration, intraperitoneal administration, intramuscularadministration, intranodal administration, intracoronary administration,intraarterial administration (e.g., into a carotid artery), subcutaneousadministration, transdermal delivery, intratracheal administration,subcutaneous administration, intraarticular administration,intraventricular administration, inhalation (e.g., aerosol),intracranial, intraspinal, intraocular, intranasal, oral, bronchial,rectal, topical, vaginal, urethral, pulmonary administration,impregnation of a catheter, and direct injection into a tissue. Routesuseful for deliver to mucosal tissues include, bronchial, intradermal,intramuscular, intranasal, other inhalatory, rectal, subcutaneous,topical, transdermal, vaginal and urethral routes. Combinations ofroutes of delivery can be used and in some instances, may enhance thetherapeutic effects of the composition. Particularly preferred routes ofdelivery include subcutaneous and intravenous delivery.

Ex vivo administration refers to performing part of the regulatory stepoutside of the patient, such as administering a composition of thepresent invention to a population of cells removed from a patient underconditions such that the composition contacts and/or enters the cell,and returning the cells to the patient. Ex vivo methods are particularlysuitable when the target cell type can easily be removed from andreturned to the patient.

Many of the above-described routes of administration, includingintravenous, intraperitoneal, intradermal, and intramuscularadministrations can be performed using methods standard in the art.Aerosol (inhalation) delivery can also be performed using methodsstandard in the art (see, for example, Stribling et al., Proc. Natl.Acad. Sci. USA 189:11277-11281, 1992, which is incorporated herein byreference in its entirety). Oral delivery can be performed by complexinga therapeutic composition of the present invention to a carrier capableof withstanding degradation by digestive enzymes in the gut of ananimal. Examples of such carriers include plastic capsules or tabletssuch as those known in the art.

One method of local administration is by direct injection. Directinjection techniques are particularly useful for administering acomposition to a cell or tissue that is accessible by surgery, andparticularly, on or near the surface of the body. Administration of acomposition locally within the area of a target cell refers to injectingthe composition centimeters and preferably, millimeters from the targetcell or tissue.

Various methods of administration and delivery vehicles disclosed hereinhave been shown to be effective for delivery of a nucleic acid moleculeto a target cell, whereby the nucleic acid molecule transfected the celland was expressed. In many studies, successful delivery and expressionof a heterologous gene was achieved in preferred cell types and/or usingpreferred delivery vehicles and routes of administration of the presentinvention.

In the method of the present invention, compositions can be administeredto any animal and preferably, to any member of the Vertebrate class,Mammalia, including, without limitation, primates, rodents, livestockand domestic pets. Livestock include mammals to be consumed or thatproduce useful products (e.g., sheep for wool production). Preferredmammals to protect include humans, dogs, cats, mice, rats, sheep,cattle, horses and pigs, with humans being particularly preferred.

The following examples are provided to demonstrate how the “rules”described herein can be used to create cysteine-added variants of IL-11.The examples also demonstrate the therapeutic uses of cysteine variantsof the present invention. The examples are not intended to be limiting,but only exemplary of specific embodiments of the invention.

EXAMPLES Example 1 Cloning of IL-11

IL-11 is a pleiotropic cytokine that stimulates hematopoiesis,lymphopoeisis and acute phase responses. IL-11 shares many biologicaleffects with IL-6. The amino acid sequence of human IL-11 (SEQ ID NO:17)is given in Kawashima et al. (1991) and Paul et al. (1990) bothincorporated herein by reference. IL-11 is synthesized as a precursorprotein of 199 amino acids that is cleaved to yield a mature protein of178 amino acids. There are no N-linked glycosylation sites in theprotein. IL-11 has four major alpha helices referred to as helices A-D.Relative to the amino acid sequence shown in SEQ ID NO:17, helix Aencompasses amino acids 37-56, helix B encompasses amino acids 92-112,helix C encompasses amino acids 125-147 and helix D encompasses aminoacids 173-196 Amino acids 1-21 of SEQ ID NO:17 encompass the IL-11signal sequence.

This invention provides cysteine-added variants of IL-11, whereincysteine substitutions or insertions are made in one or more of theregion proximal to the A helix (amino acids 22-36 of SEQ ID NO:17),distal to the D helix (amino acids 197-199 of SEQ ID NO:17), in the A-Bloop (amino acids 57-91 of SEQ ID NO:17), in the B-C loop (amino acids113-124 of SEQ ID NO: 17), and in the C-D loop (amino acids 148-172 ofSEQ ID NO:17). This invention also provides cysteine-added variants atthe first three or last three amino acids in any one or more of helicesA, B, C and D. Variants in which cysteine residues are added proximal tothe first amino acid of the mature protein (amino acid 22 of SEQ IDNO:17) or distal to the final amino acid of the mature protein (aminoacid 199 of SEQ ID NO:17) also are provided.

Preferred site for cysteine substitutions in the region preceding helixA of IL-11 are: P22, G23, P24, P25, P26, G27, P28, P29, R30, V31, S32,P33, D34, P35, and R36. Preferred sites for cysteine substitutions inthe A-B loop of IL-11 are: A57, A58, Q59, L60, R61, D62, K63, F64, P65,A66, D67, G68, D69, H70, N71, L72, D73, S74, L75, P76, T77, L78, A79,M80, S81, A82, G83, A84, L85, G86, A87, L88, Q89, L90, and P91.Preferred sites for cysteine substitutions in the C-D loop of IL-11 are:A148, L149, P150, Q151, P152, P153, P154, D155, P156, P157, A158, P159,P160, L161, A162, P163, P164, S165, 5166, A167, W168, G169, G170, I171,and R172. Preferred sites for cysteine substitutions in region followinghelix D of IL-11 are: T197, R198, and L199. Preferred sites for cysteinesubstitutions in the first 3 amino acids of helix A are: A37, D38, andL39. Preferred sites for cysteine substitutions in the last 3 aminoacids of helix A are: R54, Q55, and L56. Preferred sites for cysteinesubstitutions in the first 3 amino acids of helix B are: G92, V93, andL94. Preferred sites for cysteine substitutions in the last 3 aminoacids of helix B are: W110, L111, and R112. Preferred sites for cysteinesubstitutions in the first 3 amino acids of helix C are: E125, L126, andG127. Preferred sites for cysteine substitutions in the last 3 aminoacids of helix C are: 5145, R146, and L147. Preferred sites for cysteinesubstitutions in the first 3 amino acids of helix D are: A173, A174, andH175. Preferred sites for cysteine substitutions in the last 3 aminoacids of helix D are: L194, L195, and K196.

A full-length cDNA encoding IL-11 was amplified by PCR as two segmentswhich were then subsequently spliced together to generate the fulllength clone by the technique of “overlap extension” (Horton et al.,1993). One segment, encoding amino acids 1 through 147, was amplified byPCR from single-stranded cDNA derived from total RNA extracted from thehuman bladder carcinoma cell line 5637 (American Type CultureCollection, Rockville, Md.). A PCR reaction using the products of thefirst strand synthesis as template was carried out with forward primerBB265 (5>CGCAAGCTTGCCACCATGAACTG TGTTTGCCGCCTG>3; SEQ ID NO:42) andreverse primer BB273 (5>GCGGGACATCAGGAG CTGCAGCCGGCGCAG>3; SEQ IDNO:43). Primer BB265 anneals to the 5′ end of the coding sequence forthe IL-11 secretory signal sequence and the reverse primer, BB273,anneals to sequence encoding amino acids 138-147 and spans the junctionof exons 4 and 5 (McKinley et al., 1992). The ˜450 bp product of thisreaction was gel-purified and used in subsequent splicing reactions. Thesecond segment, containing DNA sequences encoding amino acids 142through 197, was amplified by PCR from human genomic DNA (STRATAGENE,San Diego, Calif.). A PCR reaction using human genomic DNA as templatewas carried out with forward primer BB272(5>CAGCTCCTGATGTCCCGCCTGGCCCTG>3; SEQ ID NO:44) and reverse primer BB274(5>AGTCTTCAGCAGCAGCAGTCCCCTCAC>3; SEQ ID NO:45). BB272 anneals tosequences encoding amino acids 142-150 and spans the junction of exons 4and 5. BB274 anneals to sequence encoding amino acids 189-197. The ˜170bp product of this reaction was gel-purified and used in subsequentsplicing reactions.

The gel purified ˜450 bp and ˜170 bp products were spliced together in aPCR reaction which included the ˜450 bp and ˜170 bp products as templateand forward primer BB265 (described above) and reverse primer BB275(5>CGCGGATCCTCCGACAGCCGAGTCTTCAGCAGCAG>3; SEQ ID NO:46). BB275 annealsto the DNA sequence encoding amino acids 191-199. The ˜620 bp product ofthis reaction was gel-purified, digested with HindIII and Bam HI andcloned into pcDNA3.1(+) vector (Invitrogen Corporation, Carlsbad,Calif.) that had been digested with Hind III and Bam HI, alkalinephosphatase treated, and gel purified. A clone with the correct DNAsequence was designated pcDNA3.1(+)::IL-11fus or pBBT298.

Example 2 Expression of IL-11 in Insect Cells

IL-11 was expressed in insect cells and secreted using the IL-11 signalsequence present in the cDNA clone. For insect cell expression, thecloned IL-11 cDNA of pBBT298 was modified at both the 5′ and 3′ ends tocreate a “flag-tagged” IL-11 cDNA. At the 5′ end, the sequence CAAA wasadded immediately upstream of the initiator ATG to enhance translation.This sequence comprises a proposed consensus translational initiationsequence for baculovirus (Ranjan and Hasnain, 1995). At the 3′ end, DNAencoding the 8 amino acid FLAG sequence(asp-tyr-lys-asp-asp-asp-asp-lys; SEQ ID NO:47), preceded by a flexiblelinker encoding the sequence: ser-gly-gly-ser-gly-gly-ser (SEQ IDNO:48), was added following amino acid 199 to provide a simplepurification system. DNA encoding the FLAG epitope was fused to theIL-11 gene. These modifications were made via PCR using oligonucleotideprimers that incorporated the desired additions to the IL-11 sequenceand the DNA sequence of this construct was confirmed. For expression inbaculovirus, the “FLAG-tagged” IL-11 cDNA was cloned into thebaculovirus transfer vector pBlueBac4.5 (Invitrogen). Purified plasmidDNAs were used to cotransfect Spodoptera frugiperda derived insect cellline Sf9 along with linearized (Bsu36 I digested) Bac-N-Blue™(Invitrogen) baculovirus DNA. The co-transfection was performedaccording to the Invitrogen “Bac-N-Blue™ Transfection Kit” protocolsusing 2×10⁶ Sf 9 cells to generate a ˜2 ml supernatant. Dilutions ofthis supernatant were assayed on Sf 9 cells at 27° C. for plaqueformation. Ten plaques were picked and each plaque was used to inoculate2.5×10⁶ Sf 9 cells in a T25 flask containing 5 ml of Grace's InsectMedia supplemented with 10% fetal bovine serum (FBS). After 5 days thesupernatants from these infected cells (the “P1” stocks) were collectedand six supernatants were tested by Western Blot for IL-11 expressionusing a polyclonal anti-IL-11 antisera obtained from R&D Systems, Inc.Alkaline phosphatase conjugated rabbit anti-goat IgG1 (Pierce Chemicalcompany) was used as the secondary antibody. Western blots weredeveloped using a NBT/BCIP development substrate (Promega Corporation,Madison, Wis.). We also constructed an IL-11 mutein in which P22, thefirst amino acid of the mature protein, is deleted (referred to as delP22). The del P22 mutant was expressed in insect cells using similarprocedures. Several plaques for both wild type IL-11 and the del P22mutant were positive for IL-11 protein expression, as judged by Westernblot. One positive supernatant for each protein was tested in the invitro IL-11 bioassay described in Example 3. Both supernatantsstimulated proliferation of the IL-11-dependent cell line in adose-dependent manner, indicating that they contained biologicallyactive IL-11 protein.

Example 3 Construction, Expression and Purification of IL-11 CysteineMuteins from Insect Cells

This example provides cysteine variants of IL-11. The novelIL-11-derived molecules of this example can be formulated and tested foractivity essentially as set forth in Example 2 above for wild-typeIL-11.

IL-11 muteins containing a single cysteine substitution were constructedin the wild type human IL-11 sequence. The IL-11 cysteine muteins wereexpressed in insect cells as described in Example 2. The IL-11 aminoacid sequence is shown in SEQ ID NO:17 of PCT Publication No. WO99/03887, incorporated herein by reference in its entirety, whichrepresents the amino acid sequence of the 199 amino acid precursorprotein, wherein amino acids 1-21 comprise the IL-11 signal sequence andare not present in the mature IL-11 protein. The following cysteinesubstitution muteins were constructed by site-directed mutagenesis andare numbered according to SEQ ID NO:17: P22C, G23C, P24C, G27C, Q151C,A158C and A162C. In addition, the inventors constructed a cysteinemutein in which a cysteine residue was added to the carboxy-terminus ofthe protein, i.e., immediately following amino acid 199. This mutein wastermed *200C.

The P22C, G23C, P24C, G27C, Q151C, A158C, A162C and *200C muteins wereexpressed in insect cells as described in Example 2. Muteins P22C, G23C,P24C, G27C, A162C and *200C were tested for biological activity versus awild type IL-11 control in an in vitro cell-line based proliferationassay. IL-11 control proteins were IL-11 prepared by us and an IL-11protein obtained from R&D Systems, Inc. Supernatants of baculovirusinfected insect cell lysates were tested in the bioassay, and the IL-11cysteine mutein or wild type IL-11 protein present in the lysate wasquantitated by a commercially available (R & D Systems) IL-11 ELISAassay. The bioassay measures IL-11-stimulated proliferation of aderivative the B9 cell line that has been adapted to proliferate inresponse to IL-11. The mouse B9 hybridoma cell line was obtained fromthe German Collection of Microorganisms and Cell Cultures (DSMZ). The B9line was passaged in IL-11 to select for a line that proliferates inresponse to IL-11 (referred to as B9-11 cells).

B9-11 cells were maintained in RPMI 1640 media supplemented with 10%FBS, 50 units/ml penicillin, 50 μg/ml streptomycin, 50 μM betamercaptoethanol and 50 ng/ml recombinant human IL-11 (R&D Systems,Inc.). For bioassays, B9-11 cells were washed and resuspended at aconcentration of 1×10⁵/ml in cell maintenance media minus IL-11. Fiftyμl (5×10³ cells) of the cell suspension was aliquotted per test well ofa flat bottom 96 well tissue culture plate. Serial 3-fold dilutions ofthe protein samples were prepared in maintenance media minus IL-11.Serial dilutions of recombinant human IL-11 (R&D Systems, Inc.) wereanalyzed in parallel. Fifty μl of the diluted protein samples were addedto the test wells and the plates incubated at 37° C. in a humidified 5%CO₂ tissue culture incubator. Protein samples were assayed in triplicatewells. After three days, 20 μl of CellTiter 96 AQueous One Solution(Promega Corporation, Madison, Wis.) was added to each well and theplates incubated at 37° C. in the tissue culture incubator for 1-4 h.Absorbance of the wells was read at 490 nm using a microplate reader.Control wells contained media but no cells. Mean absorbance values forthe triplicate control wells were subtracted from mean values obtainedfor the test wells.

In this assay, all of the IL-11 cysteine muteins tested werebiologically active, as measured by their ability to stimulateproliferation of B9-11 cells. The EC₅₀ (the concentration of proteinresulting in one half the maximal stimulation of proliferation) of themuteins ranged from indistinguishable from the EC₅₀ of the wild typeIL-11 control to within approximately 2-fold of the EC₅₀ of the wildtype control. EC₅₀s are shown in Table 1.

TABLE 1 In vitro bioactivities of insect cell-expressed IL-11 and IL-11cysteine muteins. IL-11 Protein EC₅₀ (ng/ml) Wild type IL-11 (R&DSystems, Inc.) 3.0 Wild type IL-11 (Bolder BioTechnology) 4.2 del P225.9 P22C 3.3 G23C 3.4 P24C 3.3 G27C 3.4 A162C 5.0 *200C 2.8

Example 4 Preparation and Purification of PEGylated IL-11 CysteineMuteins From Insect Cells

Wild type IL-11 and three IL-11 cysteine muteins (P24C, A162C and *200C)were purified to homogeneity from the supernatants of baculovirusinfected insect cell lysates. A positive supernatant for each isolatewas used to prepare a 500 ml high titer viral stock by inoculating a 500ml spinner flask culture of Sf 9 cells in Grace's Insect Mediasupplemented with 10% FBS. The cells were grown at 27° C. Thesupernatants from these cultures were harvested after 7 days and foundto have titers of ˜10⁸ plaque-forming-units/ml. These amplified viralstocks were subsequently used to infect 500 ml cultures for larger scaleproduction of wild type IL-11 and the cysteine muteins. 500 ml culturesof Sf 9 cells in Grace's Insect Media supplemented with 10% FBS weregrown in a spinner flasks to a titer of 1.0×10⁶/ml and then infectedwith viruses at a multiplicity of infection of 1. After 3 days thesupernatants from these cultures were clarified by centrifugation andfiltered through a 0.21 μM filter. The IL-11 proteins were purified in asingle step procedure using Anti-FLAG M2 Affinity Gel (Sigma). Theaffinity gel was washed with 3 column volumes of 0.1M glycine, pH 3.0,0.02% Tween 20 and 10% glycerol, then equilibrated with 5 column volumesof 50 mM Tris pH 7.5, 150 mM NaCl, 0.02% Tween 20 and 10% glycerol. Forwild type IL-11 the clarified baculoviral cell supernatant was adjustedto 150 mM NaCl, and the equilibrated resin was added. For IL-11 cysteinemuteins the clarified baculoviral cell supernatant was adjusted to 150mM NaCl, 5 mM cystine and the equilibrated resin was added. Batchloading was done at 4° C. overnight on a roller bottle apparatus and theresin was recovered using a Pharmacia XK 16/20 FPLC column (GEHealthcare) and washed with Tris buffer until the A280 reached baseline.The bound protein was eluted with 0.1M glycine pH 3.0, 0.02% Tween 20,10% glycerol and fractions were collected and neutralized with 1.0M TrispH 9.0. Column fractions were prepared in SDS-PAGE sample buffer withthe addition of 2% BME (beta-mercaptoethanol) when desirable andelectrophoresed on precast 14% Tris-glycine polyacrylamide gels. Thepurified IL-11 cysteine muteins were predominantly monomeric. Onnon-reducing gels a small amount of dimeric material was observed in thepurified cysteine muteins but not in purified wild type IL-11. Underreducing conditions only the monomer forms were observed, indicatingthat the dimer is formed through an intermolecular disulphide bond. Thepurified proteins were estimated to be greater than 90% pure byCoomassie Blue staining of SDS gels. The purified proteins wereconcentrated for subsequent experiments. Fractions from the anti-FLAG M2affinity column that contained most of the IL-11 proteins were pooledand loaded onto a CM S Sepharose Fast Flow (GE Healthcare) column Thebound protein was eluted with a 1 M NaCl bump.

PEGylation reactions included 200 μg of each protein, a 20× molar excessof 20 kDa PEG maleimide and a 20× molar excess of TCEP(Tris[2-carboxyethylphosphine]hydrochloride, Pierce Chemical Company).Reactions were performed at pH 8.0 at room temperature. Controlexperiments demonstrated that the IL-11 cysteine muteins needed to bereduced, at least partially, for efficient PEGylation. After 2 hours thePEGylation mixture was diluted 10× with 10 mM borate, pH 8.0, 10%glycerol, 0.02% Tween 20 (Buffer A) and loaded onto a 1 ml S-Sepharosecolumn equilibrated in Buffer A. The column was washed withequilibration buffer and bound proteins eluted by a 1 M NaCl bump in 10mM borate, pH 8.0, 10% glycerol, 0.02% Tween 20. This step served toconcentrate the material prior to separation of PEGylated and unmodifiedforms via size exclusion chromatography. A Superdex 200 10/30 column wasequilibrated with 50 mM NaPO₄ pH 7.5, 150 mM NaCl, 10% glycerol. Afterequilibration, a 0.5 ml sample containing the concentrated PEG-IL-11cysteine muteins was loaded onto the sizing column and an isocraticgradient was run. Fractions containing PEGylated proteins wereidentified by SDS-PAGE.

SDS-PAGE analysis showed that the major early peak consisted ofhomogeneous monoPEGylated IL-11 cyteine mutein. Only monoPEGylatedprotein was detected, consistent with the muteins containing a singlecysteine residue. Other later eluting fractions contained unreactedmonomer and some dimeric material formed during the PEG reaction.PEGylation efficiencies for these muteins were 60% or greater, asestimated from the chromatographic trace of the sizing column run.Fractions containing the PEGylated cysteine muteins but no unmodifiedprotein, were pooled, stored −80° C., and subsequently used inbioassays.

The purified cysteine muteins and the purified PEGylated cysteinemuteins were assayed for biological activity vs. a wild type IL-11control in the in vitro B9-11 cell-line proliferation assay. Proteinconcentrations were determined using human IL-11 ELISAs (R&D Systems,Inc.) and by Bradford analysis. All of the purified cysteine muteins andthe purified PEGylated cysteine muteins were biologically active. TheEC₅₀s of the purified cysteine muteins and the purified PEGylatedcysteine muteins ranged from indistinguishable from the EC₅₀ of the wildtype IL-11 control to within approximately 2-fold of the EC₅₀ of thewild type control. EC₅₀s of the proteins measured by ELISA were lowerthan the EC₅₀s measured by Bradford analysis.

TABLE 2 In vitro bioactivities of insect cell-expressed IL-11, IL-11cysteine muteins and PEGylated IL-11 cysteine muteins. Mean EC₅₀ ± SDMean EC₅₀ ± SD (from ELISA) ^(a) (from Bradford) ^(a) IL-11 protein(ng/mL) (ng/mL) Wild type IL-11 (R&D Systems) 4.0 ± 1.1 Not determinedWild type IL-11 (Bolder 4.9 ± 0.8 14.7 ± 2.3 BioTechnology) P24C 4.8,5.6 17, 20 20 kDa-PEG-P24C 2.0 21 A162C 6.6 ± 1.7 13.8 ± 3.5 20kDA-PEG-A162C 3.0 ± 0.6 18.6 ± 3.8 *200C 5.3 ± 1.7  8.7 ± 2.7 20kDA-PEG-*200C 1.1 ± 0.3  8.7 ± 2.2 ^(a) Mean ± standard deviation (SD)for at least 3 assays for each protein. Individual assay results areshown when there were less than 3 assays performed for a particularprotein.

For larger scale preparation of PEG-*200C for animal studies,baculovirus supernatants were clarified by centrifugation. The pH of theclarified supernatant was then adjusted to pH 7.0, centrifuged again andfiltered through a 0.45 micron filter. The IL-11*200C protein waspurified in a single step procedure using Anti-FLAG M2 Affinity Gel(Sigma). The affinity gel was washed with three column volumes of 0.1Mglycine pH 3.0, 0.02% Tween 20, 10% glycerol, then equilibrated with 5column volumes of 50 mM Tris pH 7.5, 150 mM NaCl, 0.02% Tween 20 and 10%glycerol. The washed resin was added to the clarified supernatant andbatch loaded at 4° C. overnight on a roller bottle apparatus. The resinwas collected in a Pharmacia XK column and the resin washed with theTris buffer until the A₂₈₀ reached baseline. The bound protein waseluted with 0.1 M glycine pH 3.0 and fractions collected and neutralizedwith 1.0 M Tris pH 9.2. Column fractions were analyzed by SDS-PAGE andthe fractions containing the IL-11*200C protein were pooled.

Larger scale PEGylation reactions included 1-2 mg of the IL-11 *200Cprotein, a 20× molar excess of 20 kDa maleimide PEG and a 20× molarexcess of TCEP. Reactions were performed at pH 8.0 at room temperature.After 2 hours, the PEGylation reaction mixture was diluted 10-fold with20 mM NaPO₄ pH 6.0-7.0, 0.02% Tween 20, 10% glycerol (Buffer A) andloaded onto a 1 ml Hi Trap S-Sepharose column equilibrated in Buffer A.The column was washed with equilibration buffer and the bound proteinseluted with a 20 mM NaPO₄ pH 6.0-7.0, 0.02% Tween 20, 10% glycerol, 500mM NaCl (Buffer B) step. This step served to concentrate the materialprior to separation of the PEGylated and unmodified forms of the proteinvia size exclusion chromatography. A Superdex 200 10/30 column wasequilibrated with 50 mM NaPO₄ pH 7.0, 150 mM NaCl, 10% glycerol. Afterequilibration a 0.5 ml sample was loaded onto the sizing column and anisocratic gradient was run. Column fractions were analyzed by SDS-PAGEand the fractions containing the PEGylated IL-11*200C were pooled.Similar procedures were used to prepare the IL-11 *200C protein modifiedwith a branched 40 kDa-maleimide PEG.

Example 5 Pharmacokinetics and Efficacy in Normal Rats

The inventors performed pharmacokinetic (PK) studies of the PEG-IL-11*200C proteins to compare their circulating half-lives to that of arecombinant human IL-11 product (Neumega®, American Home Products) inrats. Male Sprague-Dawley rats weighed 335-386 g at the beginning of thestudy. Both intravenous and subcutaneous pharmacokinetic data wereobtained. 20 kDa-PEG-IL-11 (*200C) and 40 kDa-PEG-II-11(*200C) wereprepared according to Example 4. For the intravenous delivery studies,rats received an intravenous bolus injection (0.1 mg/kg) of 20kDa-PEG-IL-11 (*200C), 40 kDa-PEG-IL-11 (*200C) or Neumega. Three ratswere used for each protein tested. Blood samples were drawn prior (time0) to injection of the test compounds and at 0.083, 0.5, 1, 2, 4, 10,24, 48, 72, 120, 168, 216 and 264 h post-injection. Subcutaneousdelivery studies were performed in a similar manner but the testcompounds were administered subcutaneously and blood samples were drawnat 0.5, 1, 2, 4, 10, 24, 48, 72, 120, 168, 216 and 264 h post-injection.Plasma levels of the proteins were measured using human IL-11 ELISA kits(R & D Systems, Minneapolis, Minn.). Standard curves were determined foreach protein to measure the relative sensitivity of the ELISA fordetecting the proteins.

The effect of the proteins on levels of circulating platelets in therats also was determined. Complete blood cell (CBC) analyses wereperformed on blood samples taken at 0, 72, 120, 168, 216 and 264 h postinjection. CBC analyses were performed on a Hemavet HV950FS MultispeciesHematology Analyzer. These studies demonstrated that plasma half-livesof the 20 kDa-PEG-IL-11 (*200C) and the 40 kDa-PEG-IL-11 (*200C)proteins were substantially greater than the half-life of Neumega.Tables 3 and 4 show plasma levels of these proteins as a function oftime following intravenous and subcutaneous injection, respectively.Following intravenous injection, Neumega is cleared rapidly from thecirculation and cannot be detected at 10 hours post injection. Incontrast, the PEGylated IL-11 cysteine analogs are easily detected andare present at 2 to 3 orders of magnitude higher concentration thanNeumega at 10 h post-injection. The 20 kDa-PEG-IL-11 (*200C) protein isstill detectable 24 h post injection, but is below the detection levelat 48 h post-injection. The 40 kDa-PEG-IL-11 (*200C) protein is stilldetectable up to 48 h post injection, but was below the assay detectionlimit at 72 h post-injection. The subcutaneous PK study gave similarresults. The Neumega was undetectable at 10 h post injection and wascleared much more rapidly than the PEGylated IL-11 (*200C) proteins,which were both detectable 24 h post-injection, but were below detectionlevels at 48 h post-injection.

TABLE 3 Plasma levels (expressed in pg/mL) of IL-11 (Neumega), 20kDa-PEG- IL11 (*200C), or 40 kDa-PEG-IL11 (*200C), following a singleintravenous injection of 100 μg protein/kg in Sprague-Dawley rats. Timepost- 20 kDa- 40 kDa- injection IL-11 PEG-IL11 PEG-IL11 (h) (Neumega)(*200C) (*200C) 0 0 0 0 0.083 308,708 ± 27,386  2,457,267 ± 160,3892,135,873 ± 292,238 0.5 58,735 ± 18,337 1,671,186 ± 89,915  1,478,560 ±268,546 1 5,040 ± 1,567 1,256,469 ± 78,250  1,234,776 ± 124,842 2 500 ±347  869,329 ± 90,768 1,091,975 ± 188,987 4 90 ± 17  566,519 ± 28,022 953,435 ± 146,025 10 0 177,223 ± 2,942   595,235 ± 135,782 24 0 1,120 ±971  115,064 ± 53,529 48 0 0  3,932 ± 3,488 Data are means ± SEM.

TABLE 4 Plasma levels (expressed in pg/mL) of IL-11 (Neumega), 20kDa-PEG-IL11 (*200C), or 40 kDa-PEG-IL11 (*200C), following a singlesubcutaneous injection of 100 μg protein/kg of in Sprague-Dawley rats.Time post- 20 kDa- 40 kDa- injection IL-11 PEG-IL11 PEG-IL11 (h)(Neumega) (*200C) (*200C) 0 0 0 0 0.5 10,223 ± 1,621  0 0 1 9,765 ±1,453 0 0 2 5,962 ± 2,438 541 ± 946 5,002 ± 4,327 4 3,375 ± 699  2,257 ±447  42,757 ± 38,747 10 0 18,959 ± 18,283 49,387 ± 31,482 24 0 1,248 ±1,082 13,617 ± 11,354 48 0 0 0 Data are means ± SEM.

The inventors discovered that the PEGylated IL-11 (*200C) proteins arepotent stimulators of platelet production in rats. Table 5 shows thelevels of circulating platelets over time in rats (N=3/group) thatreceived 20 kDa-PEG-IL-11 (*200C), 40 kDa-PEG-IL-11 (*200C), or Neumega(IL-11) by intravenous administration in the PK study described above. Asingle injection of Neumega did not have an effect on circulatingplatelet levels in these rats. In contrast a single injection of 20kDa-PEG-IL-11 (*200C) or 40 kDa-PEG-IL-11 (*200C) caused an increase incirculating platelet levels that is apparent at 72 hours post-injectionand peaks at 120 hours post-injection. For the 20 kDa-PEG-IL-11 (*200C)protein the peak value is about 30% over baseline at 72 h and returns tobaseline by 216 h. Injection of the 40 kDa-PEG-IL-11 (*200C) caused agreater increase in platelets, about 60% over baseline at 72 hours andthe platelet levels remain elevated until 264 hours post-injection.Similar results were seen with subcutaneous injection of the compounds(Table 6). These results demonstrate the superior potency of 20kDa-PEG-IL-11 (*200C) and 40 kDa-PEG-IL-11 (*200C), as compared toNeumega, at stimulating increases in levels of circulating platelets.These results demonstrate that a single injection of a PEGylated IL-11protein is capable of stimulating increases in circulating plateletswhereas a single injection of IL-11 (Neumega) has no effect oncirculating platelet levels.

TABLE 5 Circulating platelet counts (expressed in thousands/μL) inanimals receiving a single intravenous injection of 100 μg protein/kg ofNeumega (IL-11), 20 kDa- PEG-IL11 (* 200C), or 40 kDa-PEG-IL11 (* 200C).20 kDa- 40 kDa- Hours post- Neumega PEG IL-11 PEG IL-11 injection (h)(IL-11) (* 200C) (* 200C) 0  770 ± 111 748 ± 40 743 ± 58  72 748 ± 74868 ± 52 956 ± 52  120 711 ± 4   1048 ± 30 * 1294 ± 103 * 168 728 ± 38 924 ± 30 * 1146 ± 121 * 216 690 ± 41 770 ± 17 962 ± 69 * 264 667 ± 21738 ± 36 808 ± 63  Data are means ± SEM. p < 0.05 versus Neumega at sametime post-injection using a Student's two-tailed t-test.

TABLE 6 Circulating platelet counts (expressed in thousands/μL) inanimals receiving a single subcutaneous injection of 100 μg protein/kgof Neumega, 20 kDa-PEG-IL11 (* 200C), or 40 kDa-PEG-IL11 (* 200C). 20kDa- 40 kDa- Hours post- PEG IL11 PEG IL11 injection (h) Neumega (*200C) (* 200C) 0 719 ± 30  848 ± 120 711 ± 78 24 747 ± 41 805 ± 93 720 ±74 72 869 ± 31 1110 ± 124 848 ± 52 120 885 ± 14  1197 ± 41 *  1166 ±59 * 168 804 ± 54  1116 ± 89 *  1128 ± 82 * 216 794 ± 20 971 ± 82 887 ±71 264 718 ± 29 856 ± 79 803 ± 93 Data are means ± SEM. p < 0.05 versusNeumega at same time post-injection using a Student's two-tailed t-test.

Example 6 Efficacy of PEG-IL-11 (*200C) in Myelosuppressed Rats

The inventors found that the PEGylated IL-11 (*200C) cysteine muteinaccelerates recovery from thrombocytopenia in cyclophosphamide-treatedrats following every-other-day subcutaneous dosing. Male Sprague-Dawleyrats were obtained from Harlan Sprague-Dawley. The rats weighedapproximately 200 g at study initiation. Animals were acclimated for 8days prior to being placed on the study. On day −1, blood samples weredrawn and CBC analyses performed. Based on these results, rats wererandomized to groups of 5 according to their platelet levels. One groupof rats received subcutaneous injections of vehicle solution (Delbucco'sphosphate buffered saline) on days 1, 3, 5 and 7. A second group of ratsreceived an intraperitoneal injection of 100 mg/kg of cyclophosphamide(CPA) on day 0 and subcutaneous injections of vehicle solution on days1, 3, 5 and 7. A third group of rats received an intraperitonealinjection of 100 mg/kg of CPA on day 0 and subcutaneous injections of100 μg protein/kg of 20 kDa-PEG-IL-11 (*200C) on days 1, 3, 5 and 7. Afourth group of rats received an intraperitoneal injection of 100 mg/kgof CPA on day 0 and subcutaneous injections of 100 μg protein/kg of 40kDa-PEG-IL-11 (*200C) on days 1, 3, 5 and 7. Blood samples for CBCanalysis were obtained on days −1, 1, 3, 5, 6, 7, 8, 9, 10 & 13. Bloodsamples of approximately 50-100 μL were collected into EDTA Microvettetubes (Sarstedt) and analyzed with a Hemavet 950FS (Drew Scientific) todetermine levels of circulating platelets. The data are presented inTable 7. In animals that did not receive CPA, but did receive injectionsof vehicle, platelet levels did not change significantly over the courseof the experiment. In contrast, in animals that received CPA followed byinjections of vehicle, platelet levels decreased from an average of1,166×10³ cells/μL on day −1 to an average of 644×10³ cells/μL on day 5.In animals that received 20 kDa-PEGylated IL-11 (*200C), the plateletlevels decreased less to an average of 909×10³ cells/μL and this nadiroccurred on day 3. By day 5 platelet levels were already increasing inthe animals in this group and by day 6 platelet levels in these animalshad returned to baseline levels. In contrast, platelet levels did notreturn to baseline levels in the CPA+ vehicle control group until day 7.Similar results were seen in rats receiving 40 kDa-PEGylated IL-11(*200C). In animals that received 40 kDa-PEGylated IL-11 (*200C), theplatelet levels decreased less to an average of 837×10³ cells/μL andthis nadir occurred on day 3. By day 5 platelet levels were alreadyincreasing in the animals in this group and by day 6 platelet levels inthese animals had returned to baseline levels. These results demonstratethe ability of the PEGylated IL-11 (*200C) protein to reduce theseverity of thrombocytopenia and to accelerate the recovery to normalplatelet levels in myelosuppressed animals The compound could alsosimilarly be used to reduce the severity of thrombocytopenia and toaccelerate the recovery to normal platelet levels in animals or humansrendered thrombocytopenic as result of other chemical treatments,radiological treatments, disease, or idiopathic causes. Similarexperiments can be performed using different dosing regimens such asevery day, every third day and a single injection of the PEG-IL-11proteins.

TABLE 7 Circulating platelet counts (expressed in thousands/μL) inanimals receiving every other day subcutaneous injections of vehiclesolution or 100 μg protein/kg of 20 kDa- PEG-IL11 (* 200C) or 40kDa-PEG-IL11 (* 200C). CPA + 20 CPA + 40 Days post- Vehicle CPA +kDa-PEG-IL11 kDa-PEG-IL11 injection (no CPA) Vehicle (* 200C) (* 200C)−1 1161 ± 38 1166 ± 40 1167 ± 30  1168 ± 53  1 1197 ± 42 1197 ± 41 1172± 47  1183 ± 13  3 1198 ± 41  847 ± 58 909 ± 34  837 ± 17  5 1180 ± 46 644 ± 74  919 ± 91 *  947 ± 67 * 6 1197 ± 80  885 ± 59 1257 ± 29 * 1336 ± 106 * 7 1226 ± 48 1201 ± 41 1642 ± 44 * 1721 ± 36 * 8 1183 ± 201637 ± 21 1940 ± 58 * 2109 ± 19 * 9  1150 ± 119 1834 ± 25 2346 ± 69 * 2263 ± 104 * 10 1205 ± 54 1714 ± 45  2404 ± 129 * 2624 ± 54 * 13 1069 ±44 1324 ± 48 1679 ± 38 * 1670 ± 39 * Control animals did not receive CPAbut did receive every other day subcutaneous injections of vehiclesolution. Data are means ± SEM. * p < 0.05 versus CPA-treated ratsreceiving vehicle at same time post-injection using a Student'stwo-tailed t-test.

Example 7 Construction of a Synthetic IL-11 Gene for E. coli Expression

The IL-11 coding sequence contains a large number of codons that do notoccur frequently in E. coli proteins that are expressed at high levels.The presence of significant numbers of these so-called “rare codons” inthe coding sequences of heterologous proteins expressed in E. coli candramatically reduce expression levels. Therefore we constructed a“synthetic” IL-11 gene by oligonucleotide assembly in which only E.coli—preferred codons were employed. To facilitate cloning the gene wasconstructed with a Mlu I recognition site at its 5′ end and an Eco RIrecognition site at its 3′ end. The synthetic IL-11 gene was digestedwith Mlu I-Eco RI and ligated into a derivative of plasmid pUC18 thatwas digested with Mlu I and Eco RI and treated with calf intestinalphosphatase. The resulting plasmid was termed pBBT375. We constructedthe IL-11 synthetic sequence so that the initial proline residue (P22)of the mature protein was deleted. The sequence of this synthetic IL-11del P22 gene is given in SEQ ID NO:51. SEQ ID NO:51 encodes a syntheticIL-11 amino acid sequence represented herein by SEQ ID NO:52. Thissynthetic gene was used to express IL-11 and IL-11 cysteine muteins inthe E. coli cytoplasm and in the intein fusion protein system (NewEngland BioLabs, Beverly, Mass.) described in Examples 8 and 9.

Example 8 Cytoplasmic Expression of IL-11 and IL-11 Cysteine Muteins inE. coli

IL-11 del P22 was expressed in the E. coli cytoplasm by inserting thesynthetic IL-11 gene into expression vector pET21a+ (Novagen). Thisvector utilizes the phage T7 promoter and requires that the plasmid beinserted into an E. coli strain containing the T7 RNA polymerase, suchas BL21 (DE3) (Novagen). A translational coupler sequence was createdusing synthetic oligonucleotides and was incorporated at the 5′ end ofthe gene to facilitate initiation of translation. In addition, DNAencoding an initiator methionine residue was added to DNA encoding theN-terminus of the mature IL-11 del P22 protein. Similar procedures couldbe used to express IL-11 containing P22.

In addition to the del P22 construct, we made an IL-11 mutant thatdeleted amino acid residues 22 through 26 (deletes PGPPP; referred to asIL-11 del 22-26). We also made an IL-11 mutant that deleted amino acidresidues 22 through 29 (deletes PGPPPGPP; referred to as IL-11 del22-29). Each deletion mutant was made by PCR using mutagenicoligonucleotides (Scharf, 1990).

After confirming that the DNA sequences were correct, the threeN-terminal deletion mutants were incorporated into the structural genefor IL-11 using standard recombinant DNA methods. The IL-11 genes werethen subcloned into vector pET21a+. The plasmids were transformed intoE. coli strain BL21 (DE3), and expression of the IL-11 deletion variantswere induced with IPTG. We found that expression of IL-11 del P22 wasundetectable when a total E. coli cell lysate of this strain wasanalyzed by SDS-PAGE and stained with Coomassie Blue. However, bothIL-11 del 22-26 and IL-11 del 22-29 did express well as judged bySDS-PAGE of total E. coli cell lysates.

The inventors hypothesized that the string of 3 prolines near theN-terminus (P24, P25, and P26) was inhibiting translation. This theorywas tested by constructing additional N-terminal mutants that deletedamino acids 22-23 (referred to as IL-11 del 22-23), or deleted aminoacids 22-24 (referred to as IL-11 del 22-24), or deleted amino acids22-25 (referred to as IL11 del 22-25). The mutant genes were constructedby site directed mutagenesis and cloned into pET21a+. The resultingplasmids were transformed into BL21 (DE3) for expression studies. Basedon SDS-PAGE analysis of total cell lysates, the inventors found thatexpression of IL-11 del 22-23 was undetectable, IL-11 del 22-24 wasexpressed at a moderate level, and IL-11 del 22-25 expressed well. Thesedata indicate that deleting amino acids 22-24 or 22-25 improvesexpression of IL-11 proteins in E. coli.

The inventors also constructed three cysteine substitution mutants inthe IL11 del P22 gene. These mutants were IL-11 P24C/del P22, IL-11P25C/del P22, and IL-11 P26C/del P22. The IL-11 P24C/del P22 expressedwell, whereas IL-11 P25C/del P22 and IL-11 P26C/del P22 exhibitedmoderate expression. The data indicate that deleting any or all of P24,P25 or P26, or substituting non-proline amino acids for these prolineresidues improves expression of IL-11 proteins in E. coli, andpotentially in other host cells and organisms. Non-proline amino acidsthat may be substituted for P24, P25 and P26 include alanine, cysteine,serine, threonine, glycine, asparagine, glutamine, aspartic acid,glutamic acid, leucine, isoleucine, valine, phenylalanine, tryptophan,histidine, lysine, methionine, arginine and tyrosine. Other non-naturalamino acids known in the art also may be substituted for P24, P25 orP26. Cysteine is a preferred amino acid that can be substituted for P24,P25 or P26. The inventors also constructed an IL-11 mutant that combinesthe del 22-29 and *200C mutations (IL-11 del 22-29 *200C).

For expression and purification of the IL-11 proteins, cultures weregrown at 37° C., induced with 0.5 mM IPTG and grown for an additional 3h. Cells were pelleted by centrifugation and stored at −20° C. untilfurther processing. Cells from a total of 1.2 L were resuspended in 10mM Tris, 1 mM EDTA, 0.1% Tween-20, 1 mM TCEP at pH 7.5 and mechanicallylysed by one pass through a NIRO homogenizer at 600 bar. The cell lysatewas centrifuged at 8000 rpm for 30 minutes, using a Beckman JLA 10.5rotor. The supernatant was collected and the pellet stored at −20° C.The supernatant containing soluble IL-11 proteins was loaded onto a 5 mlS Sepharose HP column that was pre-equilibrated in S buffer A (20 mMTris, 10% glycerol, 0.05% Tween-20, 1 mM TCEP, pH 6.0). IL-11 proteinswere eluted using a 0 to 60% gradient of S buffer B (20 mM Tris, 10%glycerol, 0.05% Tween-20, 1 mM TCEP, 1M NaCl, pH 6.0) over 20 columnvolumes. The IL-11 proteins began eluting at 120 mM NaCl. Fractions wereanalyzed using SDS-PAGE. Fractions containing the IL-11 proteins werepooled and NaCl added to a concentration of 4M. The IL-11 protein wasloaded onto a 2 ml Toyo Pearl Phenyl Sepharose column that waspre-equilibrated in phenyl buffer A (20 mM Tris, 4M NaCl, 10% glycerol,0.05% Tween-20, pH 9.0) at room temperature. The IL-11 protein waseluted using phenyl buffer B (20 mM Tris, 10% glycerol, 0.05% Tween-20,pH 9.0) and elutes at approximately 70% buffer B (300 mM NaCl).Fractions were analyzed using SDS-PAGE. Fractions containing IL-11proteins are pooled, diluted 10-fold with S buffer A and loaded onto apre-equilibrated 1 ml CM Sepharose column. Proteins were eluted using Sbuffer B in which the proteins elute at about 200 mM NaCl. Fractionswere analyzed using SDS-PAGE and fractions containing IL-11 proteinswere pooled and quantified by Bradford Assay. The IL-11 del P22/P25Cprotein prepared this way was PEGylated as follows. PEGylation wasachieved by three additions of 2-fold molar excess 20K PEG-maleimide(6-fold molar excess total) and 5-fold (15-fold total) molar excessTCEP, over a 2 hour period (every 40 minutes) at pH 7.5, roomtemperature. 80 to 90% of the IL-11 mutein was converted to themonoPEGylated form. The inventors found that multiple additions of TCEPand PEG reagent to the cysteine analogs often gave higher yields ofPEGylated protein than a single addition of TCEP and PEG reagent. ThePEGylation reaction was diluted 4× with PEG buffer A (20 mM Tris, 10%glycerol, 0.05% Tween-20, pH 6.0) and loaded onto a pre-equilibrated 1ml S Sepharose column and eluted using a 0 to 100% gradient of PEGbuffer B (20 mM Tris, 10% glycerol, 0.05% Tween-20, pH 6.0, 1M NaCl).This column step separates PEGylated protein from non-PEGylated protein.Other reducing agents including, but not limited to, dithiothreitol,BME, cysteamine, reduced glutathionine and cysteine, can be substitutedfor TCEP in the procedures described above. The PEGylation reaction alsomay be performed by exposing the IL-11 mutein to the reducing agent andthen dialyzing the reducing agent away prior to exposure of the reducedprotein to the PEG reagent.

Insoluble IL-11 proteins present in the NIRO cell pellets weresolubilized in 2 mls of 20 mM Tris, 8M urea, 50 mM cysteine, pH 7.5 andgently rocked at room temperature for 2 hours. The solution was thendiluted 10× into refolding buffer containing 20 mM Tris, 0.1% Tween-20,pH 7.5 and incubated overnight at 4° C. The refold was diluted 5× with Sbuffer A and the pH was adjusted to 6.0 and loaded onto a 1 ml SSepharose column and purified as described above. The IL-11 proteinseluting from the S column were further purified using the PhenylSepharose column followed by the CM column. The purified IL-11 proteinswere quantified by Bradford analysis. The IL-11 proteins prepared inthis manner can be PEGylated and purified as described above.

The purified cysteine muteins and the purified PEGylated del P22/P25Ccysteine mutein were assayed for biological activity vs. a wild typeIL-11 control in the B9-11 in vitro cell-line proliferation assay. Allof the purified cysteine muteins and the purified PEGylated cysteinemuteins were biologically active, as measured by their ability tostimulate proliferation of B9-11 cells (Table 8). The EC₅₀s of thepurified cysteine muteins were within 4-fold of the EC₅₀s of the wildtype IL-11 control proteins. The EC₅₀ of the purified PEGylated delP22/P25C cysteine muteins was within 3-fold of the EC₅₀ of the wild typeIL-11 control proteins.

TABLE 8 In vitro bioactivities of IL-11 cysteine muteins and PEGylatedcysteine muteins in the B9-11 cell proliferation assay. Mean EC₅₀ ± SD(from Bradford) ^(a) IL-11 protein (ng/mL) Wild type IL-11 (Neumega) 3.2± 0.94 IL-11 del 22-29 5.4 ± 0.46 IL-11 del 22-26 2.4 ± 0.75 IL-11 del22-29 *200C 12.5 ± 0.71  IL-11 del P22/P25C 3.9 ± 0.97 20 kDA-PEG delP22/P25C 7.1 ± 2.5  IL-11 del P22/P24C 4.8 ± 0.28 ^(a) Mean ± SD for atleast 3 assays for each protein. All proteins were purified from thesoluble fraction of the Niro cell lysates except the IL-11 del 22-29*200C protein, which was purified from the Niro cell pellet.

Example 9 Expression of IL-11 and IL-11 Cysteine Muteins in E. coliusing the Intein system

The expression vector pTYB11 (New England Biolabs) contains a chitinbinding domain (CBD) flanked by a yeast intein sequence. The multiplecloning site is positioned to allow one to fuse a protein of interest tothe intein. After expression, the fusion product is bound to a chitinaffinity column, and the protein of interest is cleaved from the fusionprotein and eluted by incubating the column in a reducing agent such asdithiothreitol (DTT). Other reducing agents including, but not limitedto, BME, TCEP, cysteamine, reduced glutathionine and cysteine, can besubstituted for DTT. The protein of interest can be recovered withoutany non-native residues attached to its N-terminus.

The synthetic IL-11 del P22 gene was cloned into pTYB11 as follows. ASacI-EcoRI fragment encoding residues 38 to 199 and the terminationcodon of IL-11 del P22 was cloned from pBBT375 into pUC19 cut with Sac Iand Eco RI, producing the plasmid pBBT760.

The 5′ region of the synthetic IL-11 gene was PCR-amplified from pBBT375using primers BB915 (5′ TGCTCTAGAGCTCTTCCAACGGTCCGCCGCCGGGT; SEQ IDNO:49) and BB900 (5′ CCTAGGGAGCTCAGCACGCGG; SEQ ID NO:50). The resultingPCR fragment was digested with Xba I and Sac I, and cloned into pBBT760digested with the same restriction enzymes and phosphatase-treated,yielding plasmid pBBT782. After confirming the sequence of thePCR-generated region of the IL-11 gene, pBBT782 was digested with Sap Iand Eco RI. The released ˜550 bp fragment was gel purified and insertedinto pTYB11 that had been similarly digested. The resulting plasmid,pBBT785, was transformed into the E. coli strain ER2566 (New EnglandBioLabs), and the derived strain, BOB991 was induced with IPTG toexpress the CBD-Intein-IL-11 fusion. After a 5 hr induction at 25° C.,cells were collected by centrifugation, and a portion of the cell pelletwas analyzed by SDS-PAGE. A protein with a mobility expected for aCBD-Intein-IL-11 fusion (76.4 kDa) was observed in total cell lysate ofBOB991 that was not seen in the isogenic strain lacking the IL-11 gene.Further, Western blot analysis of the BOB991 lysate showed that theputative CBD-Intein-IL-11 fusion reacted with a commercial anti-IL-11antibody (R&D Systems).

The remaining cells were mechanically disrupted, and the soluble portionof the lysate was passed through a chitin affinity column, whichspecifically bound the CBD-Intein-IL-11 fusion. After washing withcolumn buffer (20 mM Tris, pH 8.0, 0.5M NaCl, 1 mM EDTA, 0.1% Tween-20),the chitin column was then flushed with 3 column volumes of the columnbuffer containing 50 mM DTT. The flow to the column was then stopped,and the column was left overnight at room temperature. During thisovernight incubation, the bond fusing IL-11 to the intein is cleaved.IL-11 was then eluted from the column with 5 column volumes of columnbuffer. Column fractions were analyzed by non-reducing SDS-PAGE and thefractions containing IL-11 were pooled. The cleaved IL-11 had anapparent molecular weight of approximately 24 kDa by non-reducingSDS-PAGE. In some cases the IL-11 protein was further purified using anS-Sepharose column. The chitin pool was diluted 1:10 with buffer A (20mM MES, pH 6.0, 10% glycerol, 0.1% Tween-20) and loaded onto a 1 mlS-Sepharose HiTrap column (GE Healthcare) equilibrated in Buffer A. Thebound proteins were eluted with a linear salt gradient from 0-50% BufferB (Buffer A containing 1M NaCl). Column fractions were analyzed bynon-reducing SDS-PAGE and the fractions containing IL-11 were pooled.Protein concentrations were determined using a Bradford assay (Bio-RadLaboratory). In some cases it may be preferable to pass the pooled IL-11proteins isolated from the chitin column through a Q-sepharose column toremove E. coli endotoxin prior to the S-Sepharose column step.Conditions can be chosen such that IL-11 does not bind the Q-Sepharosecolumn and elutes in the flow-though, whereas endotoxin does bind theQ-Sepharose column.

Similar procedures were used to express the following IL-11 del P22cysteine muteins: P24C, P25C, D69C, L72C, E123C and *200C. Similarprocedures can be used to express and purify other IL-11 cysteinemuteins, and IL-11 variants containing amino acid substitutions, aminoacid deletions, amino acid additions, amino acid insertions, and IL-11fusion proteins.

Example 10 PEGylation of the E. coli-Expressed IL-11 Cysteine Muteins

Aliquots of 200 to 300 μg of the purified IL-11 cysteine muteinsprepared as described in Example 9 were incubated with a 10-fold molarexcess of TCEP and a 10-fold molar excess of 20 kDa-maleimide-PEG(Nektar, Inc., Huntsville, Ala.). Approximately 3.3-fold molar excess of20 kDa-maleimide was added to the reaction three times for every 40minutes (at 0, 40, 80 min). After a total of 2 h incubation at roomtemperature, the PEGylation mixture was diluted 10× with Buffer A (20 mMMES, pH 6.0, 10% glycerol, 0.1% Tween-20). This pool was loaded onto a 1ml S-Sepharose HiTrap column (GE Healthcare) equilibrated in Buffer A.The bound proteins were eluted with a linear salt gradient from 0-50%Buffer B (Buffer A containing 1M NaCl). Column fractions were analyzedby non-reducing SDS-PAGE. The PEGylated IL-11 cysteine muteins eluted atapproximately 100-200 mM NaCl. Fractions containing purified 20 kDaPEG-IL-11 cysteine mutein protein were pooled. The IL-11 cysteinemuteins modified with a 10 kDa PEG-maleimide and a 40 kDa PEG-maleimidewere prepared using this same protocol.

The IL-11 del P22/*200C protein also was PEGylated using the followingprocedure. Aliquots of 1 mg of the purified IL-11 *200C mutein preparedas described in Example 9 were incubated with a 20-fold molar excess ofTCEP (Pierce Chemical Company) and a 20-fold molar excess of 20kDa-maleimide-PEG (Nippon Oil and Fat, NOF). After a 2 h incubation atroom temperature, the PEGylation mixture was diluted 10× with Buffer A(20 mM MES, pH 6.0, 10% glycerol, 0.1% tween-20) and loaded onto a 1 mlS-Sepharose HiTrap column (GE Healthcare) equilibrated in Buffer A. Thebound proteins were eluted with a linear salt gradient from 0-25% BufferB (Buffer A containing 1M NaCl). Column fractions were analyzed bynon-reducing SDS-PAGE. The mono-PEGylated IL-11 del P22 *200C proteineluted at approximately 140 mM NaCl. Fractions containing purifiedmono-PEGylated IL-11 del P22 *200C protein were pooled. The IL-11 delP22 *200C protein modified with a 30 kDa PEG-maleimide (NOF) and a 40kDa branched PEG-maleimide (Nektar, Inc.) were prepared using this sameprotocol. Similar procedures can be used to prepare PEGylatedderivatives of other IL-11 cysteine muteins.

The purified IL-11 cysteine muteins and the purified PEGylated cysteinemuteins were assayed for biological activity vs. a wild type IL-11control in the B9-11 in vitro cell-line proliferation assay. All of thepurified cysteine muteins and the purified PEGylated cysteine muteinswere biologically active, as measured by their ability to stimulateproliferation of B9-11 cells. The EC₅₀s of the purified cysteine muteinsranged from indistinguishable from the EC₅₀ of the wild type IL-11control to approximately 4.5-fold higher than the EC₅₀ of the wild typecontrol. The EC₅₀s of the purified PEGylated cysteine muteins rangedfrom approximately 3-fold to 6-fold higher than wild type IL-11.

TABLE 9 In vitro bioactivities of intein-expressed IL-11 cysteinemuteins and PEGylated IL-11 cysteine muteins in the B9-11 cellproliferation assay. Mean EC₅₀ ± SD IL-11 protein (ng/mL) Wild typeIL-11 (Neumega) 3.2 ± 0.94 IL-11 del P22 3.3 ± 0.64 P24C/del P22 12.0 ±3.5  20 kDa-PEG P24C/del P22 11.3 ± 6.4  P25C/del P22 2.3 ± 0.10 20kDA-PEG P25C/del P22 16 ± 4.0  D69C/del P22 7.7 ± 0.14 L72C/del P22 13.5± 2.1  E123C/del P22 6.9 ± 0.21 *200C/del P22 3.8 ± 1.3  20kDa-PEG-*200C/del P22 9.1 ± 2.6  30 kDa-PEG-*200C/del P22 9.9 ± 1.9  40kDa-PEG-*200C/del P22 24 ± 5.6  Protein concentrations were determinedfrom Bradford analysis.

Example 11 Efficacy of PEGylated E. coli-Expressed IL-11 CysteineMuteins in Normal Rats

The inventors determined whether a single subcutaneous injection ofIL-11 *200C/del P22 modified with 20 kDa-, 30 kDa- and 40 kDa-PEGs, andprepared as described in Example 10, stimulated production of plateletsin rats. Experiments were performed as described in Example 5. Ratsreceived a single bolus injection of the PEG-IL-11 proteins, Neumega(IL-11) or placebo (PBS). Three rats were used for each protein tested.CBC analyses were performed on blood samples taken on day 0, 1, 3, 5, 7,9 and 11 post injection. CBCs were performed on a Hemavet HV950FSMultispecies Hematology Analyzer.

As shown in Table 10, a single injection of placebo or Neumega did nothave an effect on circulating platelet levels in these rats. Incontrast, a single injection of 20 kDa-PEG-IL-11 (*200C/del P22), 30kDa-PEG-IL-11 (*200C/del P22) or 40 kDa-PEG-IL-11 (*200C/del P22) causedan increase in circulating platelet levels that is apparent at 3 dayspost-injection and peaks at 5-7 days post-injection. These resultsdemonstrate the superior potency of 20 kDa-PEG-IL-11 (*200C/del P22), 30kDa-PEG-IL-11 (*200C/del P22) and 40 kDa-PEG-IL-11 (*200C/del P22), ascompared to Neumega, at stimulating increases in levels of circulatingplatelets. These results demonstrate that a single injection of aPEGylated IL-11 protein is capable of stimulating increases incirculating platelets whereas a single injection of unPEGylated IL-11(Neumega) has no effect on circulating platelet levels.

TABLE 10 Circulating platelet counts (expressed in thousands/μL) inanimals receiving a single subcutaneous injection of placebo (PBS) or100 μg protein/kg of Neumega (IL-11), 20 kDa-PEG-IL11 (*200C/del P22),30 kDa-PEG-IL11 (*200C/del P22), or 40 kDa-PEG-IL11 (*200C/del P22).Neumega 20 kDa-PEG 30 kDa-PEG 40 kDa-PEG Day Placebo (IL-11) (*200C/delP22) (*200C/del P22) (*200C/del P22) 0 1116 ± 61 1107 ± 41 1102 ± 781115 ± 42  1057 ± 28    1 1026 ± 25  915 ± 47  878 ± 21 887 ± 31 998 ±23  3  976 ± 25 1058 ± 64   1166 ± 21 ^(a)  1144 ± 46 ^(a) 1160 ± 31^(a ) 5 1002 ± 36  968 ± 76    1425 ± 49 ^(a, b)  1377 ± 96 ^(a, b) 1320± 58 ^(a, b) 7 1091 ± 35 1092 ± 47 1275 ± 74  1347 ± 87 ^(a) 1410 ± 64^(a, b) 9 1085 ± 49 1161 ± 32 1208 ± 54 1223 ± 61  1215 ± 49    11 1131± 45 1011 ± 55   1340 ± 92 ^(b) 1156 ± 124 1245 ± 8^(b)    Data aremeans ± SEM. ^(a) p < 0.05 versus placebo at same time post-injectionusing a Student's two-tailed t-test. ^(b) p < 0.05 versus Neumega atsame time post-injection using a Student's two-tailed t-test.

Example 12 Efficacy of PEG-IL-11 (del 22/*200C) in Myelosuppressed Rats

We determined whether the PEGylated IL-11 (*200C/del P22) cysteinemutein prepared as described in Example 11, could accelerate recoveryfrom thrombocytopenia in cyclophosphamide-treated rats followingevery-other-day subcutaneous dosing. Male Sprague-Dawley rats wereobtained from Harlan Sprague-Dawley. The rats weighed approximately220-240 g at study initiation. On day −1, blood samples were drawn andCBC analyses performed. Based on these results, rats were randomized togroups of 5 according to their platelet levels. One group of ratsreceived subcutaneous injections of vehicle solution (phosphate bufferedsaline) on days 1, 3, 5 and 7. A second group of rats received anintraperitoneal injection of 100 mg/kg of cyclophosphamide (CPA) on day0 and subcutaneous injections of vehicle solution on days 1, 3, 5 and 7.A third group of rats received an intraperitoneal injection of 100 mg/kgof CPA on day 0 and subcutaneous injections of 100 μg protein/kg of 20kDa-PEG-IL-11 (*200C/del 22) on days 1, 3, 5 and 7. A fourth group ofrats received an intraperitoneal injection of 100 mg/kg of CPA on day 0and subcutaneous injections of 100 μg protein/kg of 30 kDa-PEG-IL-11(*200C/del P22) on days 1, 3, 5 and 7. A fifth group of rats received anintraperitoneal injection of 100 mg/kg of CPA on day 0 and subcutaneousinjections of 100 μg protein/kg of 40 kDa-PEG-IL-11 (*200C/del P22) ondays 1, 3, 5 and 7. A sixth group of rats received an intraperitonealinjection of 100 mg/kg of CPA on day 0 and subcutaneous injections of100 μg protein/kg of Neumega (IL-11) on days 1, 3, 5 and 7. Bloodsamples for CBC analysis were obtained on days −1, 1, 3, 5, 6, 7, 8, 9,10 & 13. Certain of the day 3 blood samples were lost. Blood samples ofapproximately 50-100 μL were collected into EDTA Microvette tubes(Sarstedt) and analyzed with a Hemavet 950FS (Drew Scientific) todetermine levels of circulating platelets. The data are presented inTable 11. In animals that did not receive CPA, but did receiveinjections of vehicle, platelet levels did not change significantly overthe course of the experiment. In contrast, in animals that received CPAfollowed by injections of vehicle, platelet levels decreased from anaverage of 1,017×10³ cells/μL on day −1 to an average of 306×10³cells/μL on day 5. In animals that received 20 kDa-PEGylated IL-11(*200C/del P22), the platelet levels decreased less to an average of362×10³ cells/μL on day 5. Platelet levels in these animals returned tobaseline levels by day 7. Similar results were seen in rats receiving 30kDa-PEGylated IL-11 (*200C/del P22) and 40 kDa-PEGylated IL-11(*200C/del P22). In animals that received these PEGylated IL-11(*200C/del P22) proteins, platelet levels reached a nadir on day 5 andwere back to normal pre-dose levels by day 7. In contrast, plateletlevels did not return to baseline levels in the CPA+ vehicle controlgroup until day 8. Platelet levels in animals receiving Neumega also didnot return to baseline pre-dose levels until day 8. These resultsdemonstrate the ability of the PEGylated IL-11 (*200C/del P22) proteinsto reduce the severity of thrombocytopenia and to accelerate therecovery to normal platelet levels in myelosuppressed animals. Thecompound could also similarly be used to reduce the severity ofthrombocytopenia and to accelerate the recovery to normal plateletlevels in animals or humans rendered thrombocytopenic as result of otherchemical treatments, radiological treatments, disease, drug treatmentsor idiopathic causes. Similar experiments can be performed usingdifferent dosing regimens such as every day, every third day and asingle injection of the PEG-IL-11 proteins.

TABLE 11 Circulating platelet counts (expressed in thousands/μL) inanimals receiving every other day subcutaneous injections of vehiclesolution or 100 μg protein/kg of Neumega (IL-11), 20 kDa-PEG-IL11(*200C/del P22), 30 kDa-PEG-IL11 (*200C/del P22) or 40 kDa-PEG-IL11(*200C/del P22). CPA + 20 CPA + 30 CPA + 40 Vehicle CPA + kDa-PEGkDa-PEG kDa-PEG Day (no CPA) Vehicle Neumega *200C *200C *200C −1 1012 ±71 1017 ± 88  1025 ± 68 1025 ± 70 1012 ± 80 1011 ± 69 1 1002 ± 38 890 ±58  929 ± 39 1070 ± 55  907 ± 45  988 ± 30 3 1006 ± 62 688 ± 32 — — — —5 1010 ± 48 306 ± 43  354 ± 19  362 ± 41  318 ± 12  376 ± 16 6  988 ± 21431 ± 92  562 ± 38  565 ± 51  570 ± 40  651 ± 25 7 1149 ± 42 753 ± 77 899 ± 42 1153 ± 68 1144 ± 45 1134 ± 72 8  989 ± 47 1165 ± 59  1133 ± 591175 ± 44 1206 ± 77 1374 ± 13 9  992 ± 35 1210 ± 64  1205 ± 57 1329 ± 781419 ± 21 1502 ± 73 10  995 ± 55 1255 ± 146 1178 ± 94  1843 ± 121 1770 ±77 1883 ± 50 13 1022 ± 31 1469 ± 98  1363 ± 73 1549 ± 88  1543 ± 1041411 ± 57 Control animals did not receive CPA but did receive everyother day subcutaneous injections of vehicle solution. Data are means ±SEM.

Example 13 Cysteine Analogs can be Constructed at any Position ofInterest in IL-11

Using the techniques and procedures disclosed in these Examples one ofordinary skill in the art could produce additional cysteine variants ofIL-11 and purified PEGylated forms of cysteine variants of IL-11. Thepresent inventor has determined that cysteine residues can potentiallybe substituted for any amino acid in IL-11. Cysteine substitutionmuteins can be prepared as described in the Examples and tested in theB9-11 cell proliferation assay to confirm they are biologically active.Biologically active IL-11 cysteine muteins can be reacted with cysteinereactive moieties such as cysteine reactive PEGs using proceduresdescribed in the Examples. The purified PEGylated cysteine muteins canbe tested in the B9-11 cell proliferation assay to determine if they arebiologically active. Biologically active cysteine muteins of IL-11 canbe tested in animal models to determine if they stimulate hematopoiesis,and in particular thrombopoiesis and platelet formation, using theanimal models described in the Examples. Multiple cysteine substitutionscould also be constructed by combining two or more of the above cysteinemuteins in one protein. Muteins containing more than one added cysteinecan be used to create IL-11 proteins modified with more than one PEG.

Example 14 Other Methods for Preparing PEGylated Derivatives of IL-11and IL-11 Analogs

PEGs can be attached to a peptide or protein through a variety ofchemistries that target the amino acid side chains, the amino-terminalamino acid, the carboxy-terminal amino acid, or the sugar residues inthe case of a glycosylated protein (See reviews by Veronese, 2001;Roberts et al., 2002; Morpurgo and Veronese, 2004). One preferred routefor PEG conjugation of proteins is to use a PEG with a functional groupthat reacts with lysines and/or the N-terminal amino acid group. Theliterature describes more than a dozen such procedures (see reviews byHooftman et al., 1996; Delgato et al., 1992; and Zalipsky, 1995).Examples of amine-reactive PEGs include PEG dichlorotriazine, PEGtresylate, PEG succinimidyl carbonate, PEG benzotriazole carbonate, PEGp-nitrophenyl carbonate, PEG carbonylimidazole, PEG succinimidylsuccinate, PEG propionaldehyde, PEG acetaldehyde, and PEGhydroxysuccinimide

The mature IL-11 protein has 3 lysine residues (K63, K120 and K196) inaddition to the amino terminal amino acid available for conjugation withan amine-reactive PEG. Multiple attachments may occur if the protein isexposed to an excess amount of PEGylation reagent. Preferably, the IL-11PEG conjugate would have 1-4 PEGs attached to the protein, morepreferred would be 1-2 PEG attachments, and most preferred 1 PEGattachment. Conditions can be adjusted to limit the number ofattachments or the site of PEG attachments. The number of attachmentscan be titrated by varying the molar ratios of the PEG:Protein.Preferred ratios can be determined experimentally. A second method forvarying the number of PEG attachments is by modifying the reactionconditions. For example, the coupling can be preferentially directed tothe alpha-terminus of a protein chain by performing the reaction at a pHlower than 7 and preferably below 6.5. Above pH 8, the epsilon-NH3groups found on the lysines will be most reactive with the PEG reagent(Morpurgo and Veronese, 2004). A third approach to controlling thenumber or location of the PEG conjugates is to conduct the PEGylation inthe presence of a substrate, reversible inhibitor, binding protein orsoluble receptor such as a soluble IL-11 receptor so that the aminoacids required for activity are protected during the PEG couplingreaction. A fourth approach to controlling the number of attachmentsinvolves using a larger PEG. For example when interferon-alpha ismodified with a small linear PEG polymer, up to 11 positional isomersare present in the final mixture. When interferon-alpha is modified witha larger 40 kDa branched PEG, only four main positional isomers arepresent in the mono-PEGylated protein (Monkarsh et al., 1997, Foser etal. 2003, Bailon et al. 2003). A fifth method to control the number ofattached PEGs is to use column chromatography procedures (including butnot limited to ion exchange, size exclusion or hydrophobic interaction)to purify a IL-11 conjugate containing the desired number of PEGmolecules from a more complex IL-11-PEG mixture. A six method to controlthe number of attached PEGs is to genetically modify the protein toreduce or add lysine residues to the protein's primary sequence. Forexample IL-11 analogs can be constructed in which one or more of thelysine residues (K63, K120, K196) are changed to a non-lysine amino acidsuch as arginine. Alternatively, IL-11 muteins in which a lysine residueis substituted for a non-lysine amino acid in IL-11, or added precedingthe first amino acid of the mature protein or following the last aminoacid of the mature protein can be created using standard DNA mutagensisprocedures.

PEG-hydrazide can be used to PEGylate the carboxyl groups in presence ofN,N′-dicyclohexylcarbodiimide (DCC), or in presence of a water solublecoupling agent such as N-(-3-dimethylaminopropyl)-N′-ethylcarbodiimidehydrochloride (EDC). The carboxyl groups of a protein when activatedwith EDC at an acidic pH (pH 4.5-5) react readily with PEG-hydrazide,whereas amino groups of the protein are protonated and unreactive.

Similar to the genetically engineered cysteine mutations for sitespecific PEGylation, researchers have reported the specificincorporation of unnatural amino acids into proteins expressed in yeast(Deiters et al., 2004). Specifically para-azidophenylalanine wassubstituted into a protein at certain sites determined by thepositioning of the amber codon. The reactive group on the amino acidanalog was used in a mild [3+2] cycloaddition reaction with an alkynederivatized PEG reagent to allow for site-specific conjugation. Similarprocedures can be applied to IL-11 to create PEGylated IL-11 analogs.Preferred sites for introduction of non-natural amino acids into theIL-11 coding sequence that result in biologically active IL-11 analogsand PEGylated IL-11 analogs can be determined using the methods andassays taught in the various Examples.

Another method for PEGylation of IL-11 is the attachment of the PEGmoiety on the arginine side chain using PEG-1-3-dioxo compounds such asPEG-phenylglioxate. Other amino acids such as histidines and lysines maybe modified as well.

PEG-isocyante can be used to attach a PEG to a hydroxy group via astable urethane linkage. The disadvantage of this approach is lack ofspecificity since it is also capable of reacting with amines. Thus, thisreagent is more commonly used in PEGylation reactions involvingpolysaccharides or non-peptide drugs.

Oxidation of the carbohydrate residues or N-terminal serine or threonineis an alternative method for a site-specific PEGylation. Carbohydrateside chains can be oxidized with enzymes or chemically with sodiumperiodate to generate reactive aldehyde groups. These sites can bereacted with either PEG-hydrazide or PEG-amine to produce a reversibleSchiff's base. These linkages are then reduced with sodiumcyanoborohydride to a more stable alkyl hydrazide or in the case of theSchiff's base, a secondary amine. Multiple attachment sites aregenerated by this method but the PEG is localized on the carbohydratechain rather than on the protein.

A similar approach takes advantage of an N-terminal serine or threonine.These amino acid residues can be converted by periodate oxidation to aglyoxylyl derivative which will also react with PEG-hydrazide orPEG-amine. IL-11 analogs containing an amino-terminal serine orthreonine residue can be constructed using standard DNA mutagenesisprocedures.

Another approach for PEGylation of proteins uses the enzymetransglutaminase to modify glutamine residues so they become reactivewith alkylamine derivatives of PEG. (Sato 2002). Similar procedures canbe used to create PEGylated derivatives of IL-11.

Example 16 N-terminal PEGylation of IL-11

Purified IL-11 del P22 prepared by expression of the protein in E. coliusing the intein system was incubated at a concentration of 100 μgprotein/mL with a 5- to 400-fold molar excess of a 5 kDa-amine-reactivePEG (5 kDa-methoxy-SPA-PEG, Shearwater Corporation) at pH 6.0 or pH 6.5in 100 mM MES buffer for one hour at room temperature. Analysis of thereaction mixture by SDS-PAGE showed the presence of a mono-PEGylatedIL-11 protein at PEG:protein molar ratios above 10-20× in the pH 6.0samples. The PEG-IL-11 protein migrated with an approximate molecularmass of 33 kDa by SDS-PAGE. The mono-PEGylated IL-11 protein can bepurified by column chromatography as described in the Examples. BothmonoPEGylated and diPEGylated IL-11 protein was observed in the pH 6.5samples. MonoPEGylated protein was predominant at molar ratios above 5×.DiPEGylated protein (apparent molecular mass of 45 kDa by SDS-PAGE) wasapparent at molar ratios above 75×.

Example 17 Amine-PEGylation of IL-11

Purified IL-11 del P22 prepared by expression of the protein in E. coliusing the intein system was incubated at a concentration of 100 μgprotein/mL with a 5- to 400-fold molar excess of a 5 kDa-amine-reactivePEG (5 kDa-methoxy-SPA-PEG, Shearwater Corporation) at pH 8.0 in 100 mMMES buffer for one hour at room temperature. Analysis of the reactionmixture by SDS-PAGE showed the presence of mono-PEGylated IL-11 proteinat PEG:protein molar ratios above 5×. Analysis of the reaction mixtureby SDS-PAGE showed the presence of di-PEGylated IL-11 protein atPEG:protein molar ratios above 50-75×. The mono-PEGylated IL-11 proteinmigrated with an approximate molecular mass of 33 kDa by SDS-PAGE. Thedi-PEGylated IL-11 protein migrated with an approximate molecular weightof 45 kDa. The mono-PEGylated IL-11 protein and di-PEGylated IL-11protein can be purified by column chromatography as described in theExamples. A variety of column chromatography procedures, including butnot limited to size-exclusion column chromatography can be used toisolate mono-PEGylated protein from di-PEGylated protein.

Example 18 Additional Cysteine Muteins of IL-11

Using the methods and techniques described in the Examples above,additional cysteine muteins containing a single cysteine substitution(E38C, L39C, S74C, T77C, A114C, S117C, A148C, S165C) or cysteine muteinscontaining two different cysteine substitutions in the same region or intwo different regions (P25C/T77C; P25C/S117C; P25C/S165C; P24C/P25C;D69C/T77C; A162C/S165C), were constructed in the human IL-11 gene andwere expressed in E. coli or in an insect cell expression system. Themuteins are listed in Tables 12 and 13. The reference to positionnumbers is made with regard to the IL-11 amino acid sequence with thesignal sequence (SEQ ID NO:17).

Certain of the muteins described above were expressed in insect cellsusing a baculovirus expression system and tested for biological activityvs. a wild type IL-11 control in an in vitro cell-line basedproliferation assay. Supernatants of baculovirus infected insect celllysates were tested in the bioassay, and the IL-11 cysteine mutein orwild type IL-11 protein present in the lysate was quantitated by acommercially available (R & D Systems) IL-11 ELISA assay. Certain of thecysteine muteins were subsequently purified and quantitated using aBradford dye binding assay. Other cysteine muteins were expressed in E.coli and purified. These latter muteins also were quantitated using aBradford dye binding assay. The bioassay measures IL-11-stimulatedproliferation of a derivative the B9 cell line that has been adapted toproliferate in response to IL-11. In this assay, all of the cysteinemuteins described above (Tables 12 and 13) were biologically active. TheEC50 (the concentration of protein resulting in one half the maximalstimulation of proliferation) of the muteins ranged from within 2-foldof the EC50 of the wild type IL-11 control to greater than 14-fold or33-fold higher than the EC50 of the wild type control.

Several of the cysteine muteins listed in Table 12 and wild type IL-11were purified to homogeneity from the supernatants of baculovirusinfected insect cell lysates or from E. coli lysates, and these purifiedcysteine muteins were modified with polyethylene glycol (“PEGylated”)using techniques described in the Examples above. The PEGylated forms ofthe cysteine muteins were purified away from any unmodified material.The purified cysteine muteins and the purified PEGylated cysteinemuteins were assayed for biological activity vs. a wild type IL-11control in the in vitro cell-line proliferation assay. All of thepurified cysteine muteins and the purified PEGylated cysteine muteinswere biologically active.

TABLE 12 In vitro bioactivities of Additional Cysteine Muteins of IL-11IL-11 Added cysteine EC50 with Protein EC50 (ng/mL) location 20K-PEGIL-11 3.2 +/− 0.9 (Neumega) IL-11 3.3 +/− 0.6 S74C 44.3 +/− 13.1 A-Bloop  345 +/− 21.2 T77C 5.6 +/− 0.1 A-B loop 14.3 +/− 0.5 A114C 11.5 +/−0.6  B-C loop 19.3 +/− 1.5 S117C  12 +/− 0.8 B-C loop 13.8 +/− 0.5 A148C 8 +/− 0.4 C-D loop S165C 9.8 +/− 2  C-D loop  7.6 +/− 1.1 IL-11positions are relative to SEQ ID NO: 17

TABLE 13 In vitro bioactivities of Additional Cysteine Muteins of IL-11EC50 (ng/ml) by Assay IL-11 Protein #1 #2 #3 #4 #5 #6 #7 #8 IL-11 6.36.3 5.1 5.3 6.3 6.1 7 6 IL-11 (E38C) 7.1 7.1 IL-11 (L39C) ~200 ~200IL-11 (P25C/T77C) 6.1 6.1 IL-11(P25C/S117C) 7.1 9.1 IL-11(P25C/S165C)6.3 5.8 IL-11(P24C/P25C) 5.3 6 IL-11(D69C/T77C) 5.2 6 IL-11(A162C/S165C)6 5 IL-11 positions are relative to SEQ ID NO: 17

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All of the documents cited herein are incorporated herein by reference.

The protein analogues (i.e., the cysteine variants or muteins) disclosedherein can be used for the known therapeutic uses of the native proteinsin essentially the same forms and doses all well known in the art.

While the exemplary preferred embodiments of the present invention aredescribed herein with particularity, those having ordinary skill in theart will recognize changes, modifications, additions, and applicationsother than those specifically described herein, and may adapt thepreferred embodiments and methods without departing from the spirit ofthis invention.

What is claimed is:
 1. A method to treat an animal with a disease orcondition that can be treated by wild-type interleukin-11 (IL-11), tostimulate platelet production in an animal, or to accelerate an animal'srecovery from thrombocytopenia, comprising administering to the animalan effective amount of interleukin-11 (IL-11) cysteine mutein, whereinthe mutein comprises (a) at least one cysteine residue substituted foran amino acid in IL-11 (SEQ ID NO:17) selected from the group consistingof: P22, G23, P24, G27, and A162; (b) at least one cysteine residuesubstituted for an amino acid in IL-11 (SEQ ID NO:17) selected from thegroup consisting of: P24, P25, E38, L39, D69, L72, S74, T77, A114, S117,E123, A148, and S165, and further comprises deletion of amino acid P22in IL-11 (SEQ ID NO:17); (c) at least one non-native cysteine residuewhich has been added following the C-terminal amino acid of IL-11 (SEQID NO:17); (d) at least one non-native cysteine residue which has beenadded following the C-terminal amino acid of IL-11 (SEQ ID NO:17), andfurther comprises deletion of amino acid P22 in IL-11 (SEQ ID NO:17);(e) deletion of one or more amino acids in IL-11 (SEQ ID NO:17) selectedfrom the group consisting of: deletion of amino acid P22, deletion ofamino acids 22-26 and deletion of amino acids 22-29; or (f) at least twocysteine substitutions, wherein a cysteine residue is substituted for anamino acid in IL-11 (SEQ ID NO:17) selected from the group consisting ofP25 and T77, P25 and S117, P25 and S165, P25 and P24, D69 and T77 andA162 and S165 and further comprises deletion of amino acid P22 in IL-11(SEQ ID NO:17); and wherein the mutein has biological activity in vitroas measured by proliferation of a cell line that proliferates inresponse to IL-11.
 2. The method of claim 1, wherein thethrombocytopenia is selected from the group consisting of: (a)thrombocytopenia resulting from myelosuppressive chemotherapy; (b)thrombocytopenia resulting from other chemical treatments; (c)thrombocytopenia resulting from radiological treatments; (d)thrombocytopenia resulting from disease; (e) thrombocytopenia resultingfrom idiopathic causes; (f) thrombocytopenia resulting from drugtreatments, including interferons and ribavarin; (g) thrombocytopenia inneonates; (h) thrombocytopenia resulting from myelodysplastic syndromes;(i) thrombocytopenia resulting from aplastic anemia; and (j)thrombocytopenia resulting from cirrhosis.
 3. The method of claim 1,wherein the thrombocytopenia is thrombocytopenia resulting frommyelosuppressive chemotherapy.
 4. The method of claim 1, wherein thecysteine mutein is modified with at least one polyethylene glycol. 5.The method of claim 1, wherein the IL-11 cysteine mutein is modifiedwith a cysteine-reactive moiety.
 6. The method of claim 5, wherein thecysteine-reactive moiety is a polyethylene glycol.
 7. The method ofclaim 1, wherein the cysteine mutein is administered by a route selectedfrom the group consisting of intravenous administration, intraperitonealadministration, intramuscular administration, intranodal administration,intracoronary administration, intraarterial administration, subcutaneousadministration, transdermal delivery, intratracheal administration,intraarticular administration, intraventricular administration,inhalation, intranasal, intracranial, intraspinal, intraocular, aural,oral, pulmonary administration, impregnation of a catheter, and directinjection into a tissue.